Bacillus thuringiensis gene with coleopteran activity

ABSTRACT

The invention provides nucleic acids, and variants and fragments thereof, obtained from strains of  Bacillus thuringiensis  encoding polypeptides having pesticidal activity against insect pests, including Coleoptera. Particular embodiments of the invention provide isolated nucleic acids encoding pesticidal proteins, pesticidal compositions, DNA constructs, and transformed microorganisms and plants comprising a nucleic acid of the embodiments. These compositions find use in methods for controlling pests, especially plant pests.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/020,163, filed Jan. 10, 2008, the content of which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to naturally-occurring and recombinantnucleic acids obtained from novel Bacillus thuringiensis genes thatencode pesticidal polypeptides characterized by pesticidal activityagainst insect pests. Compositions and methods of the invention utilizethe disclosed nucleic acids, and their encoded pesticidal polypeptides,to control plant pests.

BACKGROUND OF THE INVENTION

Insect pests are a major factor in the loss of the world's agriculturalcrops. For example, Western corn rootworm feeding, black cutworm damage,or European corn borer damage can be economically devastating toagricultural producers. Insect pest-related crop loss from European cornborer attacks on field and sweet corn alone has reached about onebillion dollars a year in damage and control expenses.

Traditionally, the primary method for impacting insect pest populationsis the application of broad-spectrum chemical insecticides. However,consumers and government regulators alike are becoming increasinglyconcerned with the environmental hazards associated with the productionand use of synthetic chemical pesticides. Because of such concerns,regulators have banned or limited the use of some of the more hazardouspesticides. Thus, there is substantial interest in developingalternative pesticides.

Biological control of insect pests of agricultural significance using amicrobial agent, such as fungi, bacteria, or another species of insectaffords an environmentally friendly and commercially attractivealternative to synthetic chemical pesticides. Generally speaking, theuse of biopesticides presents a lower risk of pollution andenvironmental hazards, and biopesticides provide greater targetspecificity than is characteristic of traditional broad-spectrumchemical insecticides. In addition, biopesticides often cost less toproduce and thus improve economic yield for a wide variety of crops.

Certain species of microorganisms of the genus Bacillus are known topossess pesticidal activity against a broad range of insect pestsincluding Lepidoptera, Diptera, Coleoptera, Hemiptera, and others.Bacillus thuringiensis (Bt) and Bacillus papilliae are among the mostsuccessful biocontrol agents discovered to date. Insect pathogenicityhas also been attributed to strains of B. larvae, B. lentimorbus, B.sphaericus (Harwook, ed., ((1989) Bacillus (Plenum Press), 306) and B.cereus (WO 96/10083). Pesticidal activity appears to be concentrated inparasporal crystalline protein inclusions, although pesticidal proteinshave also been isolated from the vegetative growth stage of Bacillus.Several genes encoding these pesticidal proteins have been isolated andcharacterized (see, for example, U.S. Pat. Nos. 5,366,892 and5,840,868).

Microbial insecticides, particularly those obtained from Bacillusstrains, have played an important role in agriculture as alternatives tochemical pest control. Recently, agricultural scientists have developedcrop plants with enhanced insect resistance by genetically engineeringcrop plants to produce pesticidal proteins from Bacillus. For example,corn and cotton plants have been genetically engineered to producepesticidal proteins isolated from strains of Bt (see, e.g., Aronson(2002) Cell Mol. Life Sci. 59(3):417-425; Schnepf et al. (1998)Microbiol Mol Biol Rev. 62(3):775-806). These genetically engineeredcrops are now widely used in American agriculture and have provided thefarmer with an environmentally friendly alternative to traditionalinsect-control methods. In addition, potatoes genetically engineered tocontain pesticidal Cry toxins have been sold to the American farmer.

There remains a need for new Bt toxins with a broader range ofinsecticidal activity against insect pests, e.g., toxins which areactive against a greater variety of insects from the order Coleoptera.In addition, there remains a need for biopesticides having activityagainst a variety of insect pests and for biopesticides which haveimproved insecticidal activity.

SUMMARY OF THE INVENTION

Compositions and methods are provided for impacting insect pests. Morespecifically, the embodiments of the present invention relate to methodsof impacting insects utilizing nucleotide sequences encodinginsecticidal peptides to produce transformed microorganisms and plantsthat express a insecticidal polypeptide of the embodiments. Such pestsinclude agriculturally significant pests, such as, for example: Westerncorn rootworm e.g. Diabrotica virgifera LeConte. In some embodiments,the nucleotide sequences encode polypeptides that are pesticidal for atleast one insect belonging to the order Coleoptera.

The embodiments provide a nucleic acid and fragments and variantsthereof which encode polypeptides that possess pesticidal activityagainst insect pests (e.g. SEQ ID NO: 1 encoding SEQ ID NO: 2). Thewild-type (e.g., naturally occurring) nucleotide sequence of theembodiments, which was obtained from Bt, encodes a novel insecticidalpeptide. The embodiments further provide fragments and variants of thedisclosed nucleotide sequence that encode biologically active (e.g.,insecticidal) polypeptides.

The embodiments further provide isolated pesticidal (e.g., insecticidal)polypeptides encoded by either a naturally occurring, or a modified(e.g., mutagenized or manipulated) nucleic acid of the embodiments. Inparticular examples, pesticidal proteins of the embodiments includefragments of full-length proteins and polypeptides that are producedfrom mutagenized nucleic acids designed to introduce particular aminoacid sequences into the polypeptides of the embodiments. In particularembodiments, the polypeptides have enhanced pesticidal activity relativeto the activity of the naturally occurring polypeptide from which theyare derived.

The nucleic acids of the embodiments can also be used to producetransgenic (e.g., transformed) monocot or dicot plants that arecharacterized by genomes that comprise at least one stably incorporatednucleotide construct comprising a coding sequence of the embodimentsoperably linked to a promoter that drives expression of the encodedpesticidal polypeptide. Accordingly, transformed plant cells, planttissues, plants, and seeds thereof are also provided.

In a particular embodiment, a transformed plant can be produced using anucleic acid that has been optimized for increased expression in a hostplant. For example, one of the pesticidal polypeptides of theembodiments can be back-translated to produce a nucleic acid comprisingcodons optimized for expression in a particular host, for example a cropplant such as a corn (Zea mays) plant. Expression of a coding sequenceby such a transformed plant (e.g., dicot or monocot) will result in theproduction of a pesticidal polypeptide and confer increased insectresistance to the plant. Some embodiments provide transgenic plantsexpressing pesticidal polypeptides that find use in methods forimpacting various insect pests.

The embodiments further include pesticidal or insecticidal compositionscontaining the insecticidal polypeptides of the embodiments, and canoptionally comprise further insecticidal peptides. The embodimentsencompass the application of such compositions to the environment ofinsect pests in order to impact the insect pests.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments of the invention are drawn to compositions and methodsfor impacting insect pests, particularly plant pests. More specifically,the isolated nucleic acid of the embodiments, and fragments and variantsthereof, comprise nucleotide sequences that encode pesticidalpolypeptides (e.g., proteins). The disclosed pesticidal proteins arebiologically active (e.g., pesticidal) against insect pests such as, butnot limited to, insect pests of the order Coleoptera. Insect pests ofinterest include, but are not limited to Western corn rootworm(Diabrotica virgifera LeConte).

The compositions of the embodiments comprise isolated nucleic acids, andfragments and variants thereof, which encode pesticidal polypeptides,expression cassettes comprising nucleotide sequences of the embodiments,isolated pesticidal proteins, and pesticidal compositions. Someembodiments provide modified pesticidal polypeptides characterized byimproved insecticidal activity against Coleopterans relative to thepesticidal activity of the corresponding wild-type protein. Theembodiments further provide plants and microorganisms transformed withthese novel nucleic acids, and methods involving the use of such nucleicacids, pesticidal compositions, transformed organisms, and productsthereof in impacting insect pests.

The nucleic acids and nucleotide sequences of the embodiments may beused to transform any organism to produce the encoded pesticidalproteins. Methods are provided that involve the use of such transformedorganisms to impact or control plant pests. The nucleic acids andnucleotide sequences of the embodiments may also be used to transformorganelles such as chloroplasts (McBride et al. (1995) Biotechnology 13:362-365; and Kota et al. (1999) Proc. Natl. Acad. Sci. USA 96:1840-1845).

The embodiments further relate to the identification of fragments andvariants of the naturally-occurring coding sequence that encodebiologically active pesticidal proteins. The nucleotide sequences of theembodiments find direct use in methods for impacting pests, particularlyinsect pests such as pests of the order Coleoptera. Accordingly, theembodiments provide new approaches for impacting insect pests that donot depend on the use of traditional, synthetic chemical insecticides.The embodiments involve the discovery of naturally-occurring,biodegradable pesticides and the genes that encode them.

The embodiments further provide fragments and variants of the naturallyoccurring coding sequence that also encode biologically active (e.g.,pesticidal) polypeptides. The nucleic acids of the embodiments encompassnucleic acid or nucleotide sequences that have been optimized forexpression by the cells of a particular organism, for example nucleicacid sequences that have been back-translated (i.e., reverse translated)using plant-preferred codons based on the amino acid sequence of apolypeptide having enhanced pesticidal activity. The embodiments furtherprovide mutations which confer improved or altered properties on thepolypeptides of the embodiments. See, e.g., copending U.S. applicationSer. No. 10/606,320, filed Jun. 25, 2003, and Ser. No. 10/746,914, filedDec. 24, 2003.

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe embodiments.

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The above-defined terms are more fullydefined by reference to the specification as a whole.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues (e.g., peptide nucleic acids) having the essential nature ofnatural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to that of naturally occurring nucleotides.

As used herein, the terms “encoding” or “encoded” when used in thecontext of a specified nucleic acid mean that the nucleic acid comprisesthe requisite information to direct translation of the nucleotidesequence into a specified protein. The information by which a protein isencoded is specified by the use of codons. A nucleic acid encoding aprotein may comprise non-translated sequences (e.g., introns) withintranslated regions of the nucleic acid or may lack such interveningnon-translated sequences (e.g., as in cDNA).

As used herein, “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire nucleicacid sequence or the entire amino acid sequence of a native(non-synthetic), endogenous sequence. A full-length polynucleotideencodes the full-length, catalytically active form of the specifiedprotein.

As used herein, the term “antisense” used in the context of orientationof a nucleotide sequence refers to a duplex polynucleotide sequence thatis operably linked to a promoter in an orientation where the antisensestrand is transcribed. The antisense strand is sufficientlycomplementary to an endogenous transcription product such thattranslation of the endogenous transcription product is often inhibited.Thus, where the term “antisense” is used in the context of a particularnucleotide sequence, the term refers to the complementary strand of thereference transcription product.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The terms “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass known analogues of natural amino acids that canfunction in a similar manner as naturally occurring amino acids.

Polypeptides of the embodiments can be produced either from a nucleicacid disclosed herein, or by the use of standard molecular biologytechniques. For example, a protein of the embodiments can be produced byexpression of a recombinant nucleic acid of the embodiments in anappropriate host cell, or alternatively by a combination of ex vivoprocedures.

As used herein, the terms “isolated” and “purified” are usedinterchangeably to refer to nucleic acids or polypeptides orbiologically active portions thereof that are substantially oressentially free from components that normally accompany or interactwith the nucleic acid or polypeptide as found in its naturally occurringenvironment. Thus, an isolated or purified nucleic acid or polypeptideis substantially free of other cellular material or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

An “isolated” nucleic acid is generally free of sequences (such as, forexample, protein-encoding sequences) that naturally flank the nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated nucleic acidscan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1kb of nucleotide sequences that naturally flank the nucleic acids ingenomic DNA of the cell from which the nucleic acid is derived.

As used herein, the term “isolated” or “purified” as it is used to referto a polypeptide of the embodiments means that the isolated protein issubstantially free of cellular material and includes preparations ofprotein having less than about 30%, 20%, 10%, or 5% (by dry weight) ofcontaminating protein. When the protein of the embodiments orbiologically active portion thereof is recombinantly produced, culturemedium represents less than about 30%, 20%, 10%, or 5% (by dry weight)of chemical precursors or non-protein-of-interest chemicals.

Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

As used herein, the term “impacting insect pests” refers to effectingchanges in insect feeding, growth, and/or behavior at any stage ofdevelopment, including but not limited to: killing the insect; retardinggrowth; preventing reproductive capability; antifeedant activity; andthe like.

As used herein, the terms “pesticidal activity” and “insecticidalactivity” are used synonymously to refer to activity of an organism or asubstance (such as, for example, a protein) that can be measured by, butis not limited to, pest mortality, pest weight loss, pest repellency,and other behavioral and physical changes of a pest after feeding andexposure for an appropriate length of time. Thus, an organism orsubstance having pesticidal activity adversely impacts at least onemeasurable parameter of pest fitness. For example, “pesticidal proteins”are proteins that display pesticidal activity by themselves or incombination with other proteins.

As used herein, the term “pesticidally effective amount” connotes aquantity of a substance or organism that has pesticidal activity whenpresent in the environment of a pest. For each substance or organism,the pesticidally effective amount is determined empirically for eachpest affected in a specific environment. Similarly, an “insecticidallyeffective amount” may be used to refer to a “pesticidally effectiveamount” when the pest is an insect pest.

As used herein, the term “recombinantly engineered” or “engineered”connotes the utilization of recombinant DNA technology to introduce(e.g., engineer) a change in the protein structure based on anunderstanding of the protein's mechanism of action and a considerationof the amino acids being introduced, deleted, or substituted.

As used herein, the term “mutant nucleotide sequence” or “mutation” or“mutagenized nucleotide sequence” connotes a nucleotide sequence thathas been mutagenized or altered to contain one or more nucleotideresidues (e.g., base pair) that is not present in the correspondingwild-type sequence. Such mutagenesis or alteration consists of one ormore additions, deletions, or substitutions or replacements of nucleicacid residues. When mutations are made by adding, removing, or replacingan amino acid of a proteolytic site, such addition, removal, orreplacement may be within or adjacent to the proteolytic site motif, solong as the object of the mutation is accomplished (i.e., so long asproteolysis at the site is changed).

A mutant nucleotide sequence can encode a mutant insecticidal toxinshowing improved or decreased insecticidal activity, or an amino acidsequence which confers improved or decreased insecticidal activity on apolypeptide containing it. As used herein, the term “mutant” or“mutation” in the context of a protein a polypeptide or amino acidsequence refers to a sequence which has been mutagenized or altered tocontain one or more amino acid residues that are not present in thecorresponding wild-type sequence. Such mutagenesis or alterationconsists of one or more additions, deletions, or substitutions orreplacements of amino acid residues. A mutant polypeptide shows improvedor decreased insecticidal activity, or represents an amino acid sequencewhich confers improved insecticidal activity on a polypeptide containingit. Thus, the term “mutant” or “mutation” refers to either or both ofthe mutant nucleotide sequence and the encoded amino acids. Mutants maybe used alone or in any compatible combination with other mutants of theembodiments or with other mutants. A “mutant polypeptide” may converselyshow a decrease in insecticidal activity. Where more than one mutationis added to a particular nucleic acid or protein, the mutations may beadded at the same time or sequentially; if sequentially, mutations maybe added in any suitable order.

As used herein, the term “improved insecticidal activity” or “improvedpesticidal activity” refers to an insecticidal polypeptide of theembodiments that has enhanced insecticidal activity relative to theactivity of its corresponding wild-type protein, and/or an insecticidalpolypeptide that is effective against a broader range of insects, and/oran insecticidal polypeptide having specificity for an insect that is notsusceptible to the toxicity of the wild-type protein. A finding ofimproved or enhanced pesticidal activity requires a demonstration of anincrease of pesticidal activity of at least 10%, against the insecttarget, or at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 100%,150%, 200%, or 300% or greater increase of pesticidal activity relativeto the pesticidal activity of the wild-type insecticidal polypeptidedetermined against the same insect.

For example, an improved pesticidal or insecticidal activity is providedwhere a wider or narrower range of insects is impacted by thepolypeptide relative to the range of insects that is affected by awild-type Bt toxin. A wider range of impact may be desirable whereversatility is desired, while a narrower range of impact may bedesirable where, for example, beneficial insects might otherwise beimpacted by use or presence of the toxin. While the embodiments are notbound by any particular mechanism of action, an improved pesticidalactivity may also be provided by changes in one or more characteristicsof a polypeptide; for example, the stability or longevity of apolypeptide in an insect gut may be increased relative to the stabilityor longevity of a corresponding wild-type protein.

The term “toxin” as used herein refers to a polypeptide showingpesticidal activity or insecticidal activity or improved pesticidalactivity or improved insecticidal activity. “Bt” or “Bacillusthuringiensis” toxin is intended to include the broader class of Crytoxins found in various strains of Bt, which includes such toxins as,for example, Cry1s, Cry2s, or Cry3s.

The terms “proteolytic site” or “cleavage site” refer to an amino acidsequence which confers sensitivity to a class of proteases or aparticular protease such that a polypeptide containing the amino acidsequence is digested by the class of proteases or particular protease. Aproteolytic site is said to be “sensitive” to the protease(s) thatrecognize that site. It is appreciated in the art that the efficiency ofdigestion will vary, and that a decrease in efficiency of digestion canlead to an increase in stability or longevity of the polypeptide in aninsect gut. Thus, a proteolytic site may confer sensitivity to more thanone protease or class of proteases, but the efficiency of digestion atthat site by various proteases may vary. Proteolytic sites include, forexample, trypsin sites, chymotrypsin sites, and elastase sites.

For example, research has shown that the insect gut proteases ofLepidopterans include trypsins, chymotrypsins, and elastases. See, e.g.,Lenz et al. (1991) Arch. Insect Biochem. Physiol. 16: 201-212; andHedegus et al. (2003) Arch. Insect Biochem. Physiol. 53: 30-47. Forexample, about 18 different trypsins have been found in the midgut ofHelicoverpa armigera larvae (see Gatehouse et al. (1997) Insect Biochem.Mol. Biol. 27: 929-944). The preferred proteolytic substrate sites ofthese proteases have been investigated. See, e.g., Peterson et al.(1995) Insect Biochem. Mol. Biol. 25: 765-774.

Efforts have been made to understand the mechanism of action of Bttoxins and to engineer toxins with improved properties. It has beenshown that insect gut proteases can affect the impact of Bt Cry proteinson the insect. Some proteases activate the Cry proteins by processingthem from a “protoxin” form into a toxic form, or “toxin.” See, Oppert(1999) Arch. Insect Biochem. Phys. 42: 1-12; and Carroll et al. (1997)J. Invertebrate Pathology 70: 41-49. This activation of the toxin caninclude the removal of the N- and C-terminal peptides from the proteinand can also include internal cleavage of the protein. Other proteasescan degrade the Cry proteins. See Oppert, ibid.

A comparison of the amino acid sequences of Cry toxins of differentspecificities reveals five highly-conserved sequence blocks.Structurally, the toxins comprise three distinct domains which are, fromthe N— to C-terminus: a cluster of seven alpha-helices implicated inpore formation (referred to as “domain 1”), three anti-parallel betasheets implicated in cell binding (referred to as “domain 2”), and abeta sandwich (referred to as “domain 3”). The location and propertiesof these domains are known to those of skill in the art. See, forexample, Li et al. (1991) Nature, 305:815-821 and Morse et al. (2001)Structure, 9:409-417. When reference is made to a particular domain,such as domain 1, it is understood that the exact endpoints of thedomain with regard to a particular sequence are not critical so long asthe sequence or portion thereof includes sequence that provides at leastsome function attributed to the particular domain. Thus, for example,when referring to “domain 1,” it is intended that a particular sequenceincludes a cluster of seven alpha-helices, but the exact endpoints ofthe sequence used or referred to with regard to that cluster are notcritical. One of skill in the art is familiar with the determination ofsuch endpoints and the evaluation of such functions.

In an effort to better characterize and improve Bt toxins, strains ofthe bacterium Bt were studied. Crystal preparations prepared fromcultures of the Bt strains were discovered to have pesticidal activityagainst Western corn rootworm (see Example 1). An effort was undertakento identify the nucleotide sequences encoding the crystal proteins fromthe selected strains, and the wild-type (i.e., naturally occurring)nucleic acids of the embodiments were isolated from these bacterialstrains, cloned into an expression vector, and transformed into E coli.Depending upon the characteristics of a given preparation, it wasrecognized that the demonstration of pesticidal activity sometimesrequired trypsin pretreatment to activate the pesticidal proteins. Thus,it is understood that some pesticidal proteins require proteasedigestion (e.g., by trypsin, chymotrypsin, and the like) for activation,while other proteins are biologically active (e.g., pesticidal) in theabsence of activation.

Such molecules may be altered by means described, for example, in U.S.application Ser. No. 10/606,320, filed Jun. 25, 2003, and Ser. No.10/746,914, filed Dec. 24, 2003. In addition, nucleic acid sequences maybe engineered to encode polypeptides that contain additional mutationsthat confer improved or altered pesticidal activity relative to thepesticidal activity of the naturally occurring polypeptide. Thenucleotide sequences of such engineered nucleic acids comprise mutationsnot found in the wild type sequences.

The mutant polypeptides of the embodiments are generally prepared by aprocess that involves the steps of: obtaining a nucleic acid sequenceencoding a Cry family polypeptide; analyzing the structure of thepolypeptide to identify particular “target” sites for mutagenesis of theunderlying gene sequence based on a consideration of the proposedfunction of the target domain in the mode of action of the toxin;introducing one or more mutations into the nucleic acid sequence toproduce a desired change in one or more amino acid residues of theencoded polypeptide sequence; and assaying the polypeptide produced forpesticidal activity.

Many of the Bt insecticidal toxins are related to various degrees bysimilarities in their amino acid sequences and tertiary structure andmeans for obtaining the crystal structures of Bt toxins are well known.Exemplary high-resolution crystal structure solution of both the Cry3Aand Cry3B polypeptides are available in the literature. The solvedstructure of the Cry3A gene (Li et al. (1991) Nature 353:815-821)provides insight into the relationship between structure and function ofthe toxin. A combined consideration of the published structural analysesof Bt toxins and the reported function associated with particularstructures, motifs, and the like indicates that specific regions of thetoxin are correlated with particular functions and discrete steps of themode of action of the protein. For example, many toxins isolated from Btare generally described as comprising three domains: a seven-helixbundle that is involved in pore formation, a three-sheet domain that hasbeen implicated in receptor binding, and a beta-sandwich motif (Li etal. (1991) Nature 305: 815-821).

As reported in U.S. Pat. No. 7,105,332, and pending U.S. applicationSer. No. 10/746,914, filed Dec. 24, 2003, the toxicity of Cry proteinscan be improved by targeting the region located between alpha helices 3and 4 of domain 1 of the toxin. This theory was premised on a body ofknowledge concerning insecticidal toxins, including: 1) that alphahelices 4 and 5 of domain 1 of Cry3A toxins had been reported to insertinto the lipid bilayer of cells lining the midgut of susceptible insects(Gazit et al. (1998) Proc. Natl. Acad. Sci. USA 95: 12289-12294); 2) theinventors' knowledge of the location of trypsin and chymotrypsincleavage sites within the amino acid sequence of the wild-type protein;3) the observation that the wild-type protein was more active againstcertain insects following in vitro activation by trypsin or chymotrypsintreatment; and 4) reports that digestion of toxins from the 3′ endresulted in decreased toxicity to insects.

A series of mutations may be created and placed in a variety ofbackground sequences to create novel polypeptides having enhanced oraltered pesticidal activity. See, e.g., U.S. application Ser. No.10/606,320, filed Jun. 25, 2003, now abandoned, and Ser. No. 10/746,914,filed Dec. 24, 2003. These mutants include, but are not limited to: theaddition of at least one more protease-sensitive site (e.g., trypsincleavage site) in the region located between helices 3 and 4 of domain1; the replacement of an original protease-sensitive site in thewild-type sequence with a different protease-sensitive site; theaddition of multiple protease-sensitive sites in a particular location;the addition of amino acid residues near protease-sensitive site(s) toalter folding of the polypeptide and thus enhance digestion of thepolypeptide at the protease-sensitive site(s); and adding mutations toprotect the polypeptide from degradative digestion that reduces toxicity(e.g., making a series of mutations wherein the wild-type amino acid isreplaced by valine to protect the polypeptide from digestion). Mutationsmay be used singly or in any combination to provide polypeptides of theembodiments.

In this manner, the embodiments provide sequences comprising a varietyof mutations, such as, for example, a mutation that comprises anadditional, or an alternative, protease-sensitive site located betweenalpha-helices 3 and 4 of domain 1 of the encoded polypeptide. A mutationwhich is an additional or alternative protease-sensitive site may besensitive to several classes of proteases such as serine proteases,which include trypsin and chymotrypsin, or enzymes such as elastase.Thus, a mutation which is an additional or alternativeprotease-sensitive site may be designed so that the site is readilyrecognized and/or cleaved by a category of proteases, such as mammalianproteases or insect proteases. A protease-sensitive site may also bedesigned to be cleaved by a particular class of enzymes or a particularenzyme known to be produced in an organism, such as, for example, achymotrypsin produced by the corn earworm Heliothis zea (Lenz et al.(1991) Arch. Insect Biochem. Physiol. 16: 201-212). Mutations may alsoconfer resistance to proteolytic digestion, for example, to digestion bychymotrypsin at the C-terminus of the peptide.

The presence of an additional and/or alternative protease-sensitive sitein the amino acid sequence of the encoded polypeptide can improve thepesticidal activity and/or specificity of the polypeptide encoded by thenucleic acids of the embodiments. Accordingly, the nucleotide sequencesof the embodiments can be recombinantly engineered or manipulated toproduce polypeptides having improved or altered insecticidal activityand/or specificity compared to that of an unmodified wild-type toxin. Inaddition, the mutations disclosed herein may be placed in or used inconjunction with other nucleotide sequences to provide improvedproperties. For example, a protease-sensitive site that is readilycleaved by insect chymotrypsin, e.g., a chymotrypsin found in the berthaarmyworm or the corn earworm (Hegedus et al. (2003) Arch. InsectBiochem. Physiol. 53: 30-47; and Lenz et al. (1991) Arch. InsectBiochem. Physiol. 16: 201-212), may be placed in a Cry backgroundsequence to provide improved toxicity to that sequence. In this manner,the embodiments provide toxic polypeptides with improved properties.

For example, a mutagenized Cry nucleotide sequence can compriseadditional mutants that comprise additional codons that introduce asecond trypsin-sensitive amino acid sequence (in addition to thenaturally occurring trypsin site) into the encoded polypeptide. Analternative addition mutant of the embodiments comprises additionalcodons designed to introduce at least one additional differentprotease-sensitive site into the polypeptide, for example, achymotrypsin-sensitive site located immediately 5′ or 3′ of thenaturally occurring trypsin site. Alternatively, substitution mutantsmay be created in which at least one codon of the nucleic acid thatencodes the naturally occurring protease-sensitive site is destroyed andalternative codons are introduced into the nucleic acid sequence inorder to provide a different (e.g., substitute) protease-sensitive site.A replacement mutant may also be added to a Cry sequence in which thenaturally-occurring trypsin cleavage site present in the encodedpolypeptide is destroyed and a chymotrypsin or elastase cleavage site isintroduced in its place.

It is recognized that any nucleotide sequence encoding the amino acidsequences that are proteolytic sites or putative proteolytic sites (forexample, sequences such as NGSR, RR, or LKM) can be used and that theexact identity of the codons used to introduce any of these cleavagesites into a variant polypeptide may vary depending on the use, i.e.,expression in a particular plant species. It is also recognized that anyof the disclosed mutations can be introduced into any polynucleotidesequence of the embodiments of SEQ ID NO:1 that comprise the codons foramino acid residues that provide the native trypsin cleavage site thatis targeted for modification. Accordingly, variants of eitherfull-length toxins or fragments thereof can be modified to containadditional or alternative cleavage sites, and these embodiments areintended to be encompassed by the scope of the embodiments disclosedherein.

It will be appreciated by those of skill in the art that any usefulmutation may be added to the sequences of the embodiments so long as theencoded polypeptides retain pesticidal activity. Thus, sequences mayalso be mutated so that the encoded polypeptides are resistant toproteolytic digestion by chymotrypsin. More than one recognition sitecan be added in a particular location in any combination, and multiplerecognition sites can be added to or removed from the toxin. Thus,additional mutations can comprise three, four, or more recognitionsites. It is to be recognized that multiple mutations can be engineeredin any suitable polynucleotide sequence; accordingly, either full-lengthsequences or fragments thereof can be modified to contain additional oralternative cleavage sites as well as to be resistant to proteolyticdigestion. In this manner, the embodiments provide Cry toxins containingmutations that improve pesticidal activity as well as improvedcompositions and methods for impacting pests using other Bt toxins.

Mutations may protect the polypeptide from protease degradation, forexample by removing putative proteolytic sites such as putative serineprotease sites and elastase recognition sites from different areas. Someor all of such putative sites may be removed or altered so thatproteolysis at the location of the original site is decreased. Changesin proteolysis may be assessed by comparing a mutant polypeptide withwild-type toxins or by comparing mutant toxins which differ in theiramino acid sequence. Putative proteolytic sites and proteolytic sitesinclude, but are not limited to, the following sequences: RR, a trypsincleavage site; LKM, a chymotrypsin site; and NGSR, a trypsin site. Thesesites may be altered by the addition or deletion of any number and kindof amino acid residues, so long as the pesticidal activity of thepolypeptide is increased. Thus, polypeptides encoded by nucleotidesequences comprising mutations will comprise at least one aminoacid/change or addition relative to the native or background sequence ofSEQ ID NO:2, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 35, 38, 40, 45,47, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, or 280 or more amino acidchanges or additions. Pesticidal activity of a polypeptide may also beimproved by truncation of the native or full-length sequence, as isknown in the art.

Compositions of the embodiments include nucleic acids, and fragments andvariants thereof, that encode pesticidal polypeptides. In particular,the embodiments provide for isolated nucleic acid molecules comprisingnucleotide sequences encoding the amino acid sequence shown in SEQ IDNO: 2, or the nucleotide sequences encoding said amino acid sequence,for example the nucleotide sequence set forth in SEQ ID NO: 1, andfragments and variants thereof.

Also of interest are optimized nucleotide sequences encoding thepesticidal proteins of the embodiments. As used herein, the phrase“optimized nucleotide sequences” refers to nucleic acids that areoptimized for expression in a particular organism, for example a plant.Optimized nucleotide sequences may be prepared for any organism ofinterest using methods known in the art. See, for example, U.S.application Ser. No. 10/606,320, filed Jun. 25, 2003, now abandoned, andSer. No. 10/746,914, filed Dec. 24, 2003, which describe an optimizednucleotide sequence encoding a disclosed pesticidal protein. In thisexample, the nucleotide sequence was prepared by reverse-translating theamino acid sequence of the protein and changing the nucleotide sequenceso as to comprise maize-preferred codons while still encoding the sameamino acid sequence. This procedure is described in more detail byMurray et al. (1989) Nucleic Acids Res. 17:477-498. Optimized nucleotidesequences find use in increasing expression of a pesticidal protein in aplant, for example monocot plants of the Gramineae (Poaceae) family suchas, for example, a maize or corn plant.

The embodiments further provide isolated pesticidal (e.g., insecticidal)polypeptides encoded by either a naturally-occurring or modified nucleicacid of the embodiments. More specifically, the embodiments providepolypeptides comprising an amino acid sequence set forth in SEQ ID NO:2, and the polypeptides encoded by nucleic acids described herein, forexample those set forth in SEQ ID NO: 1, and fragments and variantsthereof.

In particular embodiments, pesticidal proteins of the embodimentsprovide full-length insecticidal polypeptides, fragments of full-lengthinsecticidal polypeptides, and variant polypeptides that are producedfrom mutagenized nucleic acids designed to introduce particular aminoacid sequences into polypeptides of the embodiments. In particularembodiments, the amino acid sequences that are introduced into thepolypeptides comprise a sequence that provides a cleavage site for anenzyme such as a protease.

It is known in the art that the pesticidal activity of Bt toxins istypically activated by cleavage of the peptide in the insect gut byvarious proteases. Because peptides may not always be cleaved withcomplete efficiency in the insect gut, fragments of a full-length toxinmay have enhanced pesticidal activity in comparison to the full-lengthtoxin itself. Thus, some of the polypeptides of the embodiments includefragments of a full-length insecticidal polypeptide, and some of thepolypeptide fragments, variants, and mutations will have enhancedpesticidal activity relative to the activity of the naturally occurringinsecticidal polypeptide from which they are derived, particularly ifthe naturally occurring insecticidal polypeptide is not activated invitro with a protease prior to screening for activity. Thus, the presentapplication encompasses truncated versions or fragments of thesequences.

Mutations may be placed into any background sequence, including suchtruncated polypeptides, so long as the polypeptide retains pesticidalactivity. One of skill in the art can readily compare two or moreproteins with regard to pesticidal activity using assays known in theart or described elsewhere herein. It is to be understood that thepolypeptides of the embodiments can be produced either by expression ofa nucleic acid disclosed herein, or by the use of standard molecularbiology techniques.

It is recognized that the pesticidal proteins may be oligomeric and willvary in molecular weight, number of residues, component peptides,activity against particular pests, and other characteristics. However,by the methods set forth herein, proteins active against a variety ofpests may be isolated and characterized. The pesticidal proteins of theembodiments can be used in combination with other Bt toxins or otherinsecticidal proteins to increase insect target range. Furthermore, theuse of the pesticidal proteins of the embodiments in combination withother Bt toxins or other insecticidal principles of a distinct naturehas particular utility for the prevention and/or management of insectresistance. Other insecticidal agents include protease inhibitors (bothserine and cysteine types), α-amylase, and peroxidase.

Fragments and variants of the nucleotide and amino acid sequences andthe polypeptides encoded thereby are also encompassed by theembodiments. As used herein the term “fragment” refers to a portion of anucleotide sequence of a polynucleotide or a portion of an amino acidsequence of a polypeptide of the embodiments. Fragments of a nucleotidesequence may encode protein fragments that retain the biologicalactivity of the native or corresponding full-length protein and hencepossess pesticidal activity. Thus, it is acknowledged that some of thepolynucleotide and amino acid sequences of the embodiments can correctlybe referred to as both fragments and mutants.

It is to be understood that the term “fragment,” as it is used to referto nucleic acid sequences of the embodiments, also encompasses sequencesthat are useful as hybridization probes. This class of nucleotidesequences generally does not encode fragment proteins retainingbiological activity. Thus, fragments of a nucleotide sequence may rangefrom at least about 20 nucleotides, about 50 nucleotides, about 100nucleotides, and up to the full-length nucleotide sequence encoding theproteins of the embodiments.

A fragment of a nucleotide sequence of the embodiments that encodes abiologically active portion of a pesticidal protein of the embodimentswill encode at least 15, 25, 30, 50, 100, 200, 300, 400, 500, 600 or 700contiguous amino acids, or up to the total number of amino acids presentin a pesticidal polypeptide of the embodiments (for example, 719 aminoacids for SEQ ID NO: 2). Thus, it is understood that the embodimentsalso encompass polypeptides that are fragments of the exemplarypesticidal proteins of the embodiments and having lengths of at least15, 25, 30, 50, 100, 200, 300, 400, 500, 600 or 700, contiguous aminoacids, or up to the total number of amino acids present in a pesticidalpolypeptide of the embodiments (for example, 719 amino acids for SEQ IDNO: 2). Fragments of a nucleotide sequence of the embodiments that areuseful as hybridization probes or PCR primers generally need not encodea biologically active portion of a pesticidal protein. Thus, a fragmentof a nucleic acid of the embodiments may encode a biologically activeportion of a pesticidal protein, or it may be a fragment that can beused as a hybridization probe or PCR primer using methods disclosedherein. A biologically active portion of a pesticidal protein can beprepared by isolating a portion of one of the nucleotide sequences ofthe embodiments, expressing the encoded portion of the pesticidalprotein (e.g., by recombinant expression in vitro), and assessing theactivity of the encoded portion of the pesticidal protein.

Nucleic acids that are fragments of a nucleotide sequence of theembodiments comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300,350, 400, 450, 500, 600, 700, 800, 1,000, 1,200, 1,300 or 1,400nucleotides, or up to the number of nucleotides present in a nucleotidesequence disclosed herein (for example, 1,406 nucleotides for SEQ ID NO:1). Particular embodiments envision fragments derived from (e.g.,produced from) a first nucleic acid of the embodiments, wherein thefragment encodes a truncated toxin characterized by pesticidal activity.Truncated polypeptides encoded by the polynucleotide fragments of theembodiments are characterized by pesticidal activity that is eitherequivalent to, or improved, relative to the activity of thecorresponding full-length polypeptide encoded by the first nucleic acidfrom which the fragment is derived. It is envisioned that such nucleicacid fragments of the embodiments may be truncated at the 3′ end of thenative or corresponding full-length coding sequence. Nucleic acidfragments may also be truncated at both the 5′ and 3′ end of the nativeor corresponding full-length coding sequence.

The term “variants” is used herein to refer to substantially similarsequences. For nucleotide sequences, conservative variants include thosesequences that, because of the degeneracy of the genetic code, encodethe amino acid sequence of one of the pesticidal polypeptides of theembodiments. Naturally occurring allelic variants such as these can beidentified with the use of well-known molecular biology techniques, suchas, for example, polymerase chain reaction (PCR) and hybridizationtechniques as outlined herein.

Variant nucleotide sequences also include synthetically derivednucleotide sequences, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a pesticidal protein ofthe embodiments, such as a mutant toxin. Generally, variants of aparticular nucleotide sequence of the embodiments will have at leastabout 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to that particularnucleotide sequence as determined by sequence alignment programsdescribed elsewhere herein using default parameters. A variant of anucleotide sequence of the embodiments may differ from that sequence byas few as 1-15 nucleotides, as few as 1-10, such as 6-10, as few as 5,as few as 4, 3, 2, or even 1 nucleotide.

Variants of a particular nucleotide sequence of the embodiments (i.e.,an exemplary nucleotide sequence) can also be evaluated by comparison ofthe percent sequence identity between the polypeptide encoded by avariant nucleotide sequence and the polypeptide encoded by the referencenucleotide sequence. Thus, for example, isolated nucleic acids thatencode a polypeptide with a given percent sequence identity to thepolypeptide of SEQ ID NO: 2 are disclosed. Percent sequence identitybetween any two polypeptides can be calculated using sequence alignmentprograms described elsewhere herein using default parameters. Where anygiven pair of polynucleotides of the embodiments is evaluated bycomparison of the percent sequence identity shared by the twopolypeptides they encode, the percent sequence identity between the twoencoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, generally at least about 75%, 80%, 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, or at least about 98%, 99% or moresequence identity.

As used herein, the term “variant protein” encompasses polypeptides thatare derived from a native protein by: deletion (so-called truncation) oraddition of one or more amino acids to the N-terminal and/or C-terminalend of the native protein; deletion or addition of one or more aminoacids at one or more sites in the native protein; or substitution of oneor more amino acids at one or more sites in the native protein.Accordingly, the term “variant protein” encompasses biologically activefragments of a native protein that comprise a sufficient number ofcontiguous amino acid residues to retain the biological activity of thenative protein, i.e., to have pesticidal activity. Such pesticidalactivity may be different or improved relative to the native protein orit may be unchanged, so long as pesticidal activity is retained.

Variant proteins encompassed by the embodiments are biologically active,that is they continue to possess the desired biological activity of thenative protein, that is, pesticidal activity as described herein. Suchvariants may result from, for example, genetic polymorphism or fromhuman manipulation. Biologically active variants of a native pesticidalprotein of the embodiments will have at least about 60%, 65%, 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to the amino acid sequence for thenative protein as determined by sequence alignment programs describedelsewhere herein using default parameters. A biologically active variantof a protein of the embodiments may differ from that protein by as fewas 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5,as few as 4, 3, 2, or even 1 amino acid residue.

The embodiments further encompass a microorganism that is transformedwith at least one nucleic acid of the embodiments, with an expressioncassette comprising the nucleic acid, or with a vector comprising theexpression cassette. In some embodiments, the microorganism is one thatmultiplies on plants. An embodiment of the invention relates to anencapsulated pesticidal protein which comprises a transformedmicroorganism capable of expressing at least one pesticidal protein ofthe embodiments.

The embodiments provide pesticidal compositions comprising a transformedmicroorganism of the embodiments. In such embodiments, the transformedmicroorganism is generally present in the pesticidal composition in apesticidally effective amount, together with a suitable carrier. Theembodiments also encompass pesticidal compositions comprising anisolated protein of the embodiments, alone or in combination with atransformed organism of the embodiments and/or an encapsulatedpesticidal protein of the embodiments, in an insecticidally effectiveamount, together with a suitable carrier.

The embodiments further provide a method of increasing insect targetrange by using a pesticidal protein of the embodiments in combinationwith at least one other or “second” pesticidal protein. Any pesticidalprotein known in the art can be employed in the methods of theembodiments. Such pesticidal proteins include, but are not limited to,Bt toxins, protease inhibitors, α-amylases, and peroxidases.

The embodiments also encompass transformed or transgenic plantscomprising at least one nucleotide sequence of the embodiments. In someembodiments, the plant is stably transformed with a nucleotide constructcomprising at least one nucleotide sequence of the embodiments operablylinked to a promoter that drives expression in a plant cell. As usedherein, the terms “transformed plant” and “transgenic plant” refer to aplant that comprises within its genome a heterologous polynucleotide.Generally, the heterologous polynucleotide is stably integrated withinthe genome of a transgenic or transformed plant such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant expression cassette.

It is to be understood that as used herein the term “transgenic”includes any cell, cell line, callus, tissue, plant part, or plant thegenotype of which has been altered by the presence of heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

As used herein, the term “plant” includes whole plants, plant organs(e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny ofsame. Parts of transgenic plants are within the scope of the embodimentsand comprise, for example, plant cells, protoplasts, tissues, callus,embryos as well as flowers, stems, fruits, leaves, and roots originatingin transgenic plants or their progeny previously transformed with a DNAmolecule of the embodiments and therefore consisting at least in part oftransgenic cells.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. The class of plants that can be used in themethods of the embodiments is generally as broad as the class of higherplants amenable to transformation techniques, including bothmonocotyledonous and dicotyledonous plants. Such plants include, forexample, Solanum tuberosum and Zea mays.

While the embodiments do not depend on a particular biological mechanismfor increasing the resistance of a plant to a plant pest, expression ofthe nucleotide sequences of the embodiments in a plant can result in theproduction of the pesticidal proteins of the embodiments and in anincrease in the resistance of the plant to a plant pest. The plants ofthe embodiments find use in agriculture in methods for impacting insectpests. Certain embodiments provide transformed crop plants, such as, forexample, maize plants, which find use in methods for impacting insectpests of the plant, such as, for example, Western corn rootworm.

A “subject plant or plant cell” is one in which genetic alteration, suchas transformation, has been effected as to a gene of interest, or is aplant or plant cell which is descended from a plant or cell so alteredand which comprises the alteration. A “control” or “control plant” or“control plant cell” provides a reference point for measuring changes inphenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-typeplant or cell, i.e., of the same genotype as the starting material forthe genetic alteration which resulted in the subject plant or cell; (b)a plant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e., with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to conditions or stimulithat would induce expression of the gene of interest; or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

One of skill in the art will readily acknowledge that advances in thefield of molecular biology such as site-specific and random mutagenesis,polymerase chain reaction methodologies, and protein engineeringtechniques provide an extensive collection of tools and protocolssuitable for use to alter or engineer both the amino acid sequence andunderlying genetic sequences of proteins of agricultural interest.

Thus, the proteins of the embodiments may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the pesticidalproteins can be prepared by introducing mutations into a syntheticnucleic acid (e.g., DNA molecule). Methods for mutagenesis and nucleicacid alterations are well known in the art. For example, designedchanges can be introduced using an oligonucleotide-mediatedsite-directed mutagenesis technique. See, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York), and the references cited therein.

The mutagenized nucleotide sequences of the embodiments may be modifiedso as to change about 1, 2, 3, 4, 5, 6, 8, 10, 12 or more of the aminoacids present in the primary sequence of the encoded polypeptide.Alternatively, even more changes from the native sequence may beintroduced such that the encoded protein may have at least about 1% or2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or even about13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%,30%, 35%, or 40% or more of the codons altered, or otherwise modifiedcompared to the corresponding wild-type protein. In the same manner, theencoded protein may have at least about 1% or 2%, or about 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, or even about 13%, 14%, 15%, 16%, 17%,18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or moreadditional codons compared to the corresponding wild-type protein. Itshould be understood that the mutagenized nucleotide sequences of theembodiments are intended to encompass biologically functional,equivalent peptides which have pesticidal activity, such as an improvedpesticidal activity as determined by antifeedant properties againstWestern corn rootworm larvae. Such sequences may arise as a consequenceof codon redundancy and functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.

One of skill in the art would recognize that amino acid additions and/orsubstitutions are generally based on the relative similarity of theamino acid side-chain substituents, for example, their hydrophobicity,charge, size, and the like. Exemplary amino acid substitution groupsthat take various of the foregoing characteristics into considerationare well known to those of skill in the art and include: arginine andlysine; glutamate and aspartate; serine and threonine; glutamine andasparagine; and valine, leucine, and isoleucine.

Guidance as to appropriate amino acid substitutions that do not affectbiological activity of the protein of interest may be found in the modelof Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl.Biomed. Res. Found., Washington, D.C.), herein incorporated byreference. Conservative substitutions, such as exchanging one amino acidwith another having similar properties, may be made.

Thus, the genes and nucleotide sequences of the embodiments include boththe naturally occurring sequences and mutant forms. Likewise, theproteins of the embodiments encompass both naturally occurring proteinsand variations (e.g., truncated polypeptides) and modified (e.g.,mutant) forms thereof. Such variants will continue to possess thedesired pesticidal activity. Obviously, the mutations that will be madein the nucleotide sequence encoding the variant must not place thesequence out of reading frame and generally will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays, such as insect-feeding assays.See, for example, Marrone et al. (1985) J. Econ. Entomol. 78: 290-293and Czapla and Lang (1990) J. Econ. Entomol. 83: 2480-2485, hereinincorporated by reference.

Variant nucleotide sequences and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different codingsequences can be manipulated to create a new pesticidal proteinpossessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, full-length codingsequences, sequence motifs encoding a domain of interest, or anyfragment of a nucleotide sequence of the embodiments may be shuffledbetween the nucleotide sequences of the embodiments and correspondingportions of other known Cry nucleotide sequences to obtain a new genecoding for a protein with an improved property of interest.

Properties of interest include, but are not limited to, pesticidalactivity per unit of pesticidal protein, protein stability, and toxicityto non-target species particularly humans, livestock, and plants andmicrobes that express the pesticidal polypeptides of the embodiments.The embodiments are not bound by a particular shuffling strategy, onlythat at least one nucleotide sequence of the embodiments, or partthereof, is involved in such a shuffling strategy. Shuffling may involveonly nucleotide sequences disclosed herein or may additionally involveshuffling of other nucleotide sequences known in the art. Strategies forDNA shuffling are known in the art. See, for example, Stemmer (1994)Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore etal. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl.Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291;and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The nucleotide sequences of the embodiments can also be used to isolatecorresponding sequences from other organisms, particularly otherbacteria, and more particularly other Bacillus strains. In this manner,methods such as PCR, hybridization, and the like can be used to identifysuch sequences based on their sequence homology to the sequences setforth herein. Sequences that are selected based on their sequenceidentity to the entire sequences set forth herein or to fragmentsthereof are encompassed by the embodiments. Such sequences includesequences that are orthologs of the disclosed sequences. The term“orthologs” refers to genes derived from a common ancestral gene andwhich are found in different species as a result of speciation. Genesfound in different species are considered orthologs when theirnucleotide sequences and/or their encoded protein sequences sharesubstantial identity as defined elsewhere herein. Functions of orthologsare often highly conserved among species.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.),hereinafter “Sambrook”. See also Innis et al., eds. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NewYork); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press,New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual(Academic Press, New York). Known methods of PCR include, but are notlimited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the sequences of theembodiments. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook.

For example, an entire sequence disclosed herein, or one or moreportions thereof, may be used as a probe capable of specificallyhybridizing to corresponding sequences and messenger RNAs. To achievespecific hybridization under a variety of conditions, such probesinclude sequences that are unique to the sequences of the embodimentsand are generally at least about 10 or 20 nucleotides in length. Suchprobes may be used to amplify corresponding Cry sequences from a chosenorganism by PCR. This technique may be used to isolate additional codingsequences from a desired organism or as a diagnostic assay to determinethe presence of coding sequences in an organism. Hybridizationtechniques include hybridization screening of plated DNA libraries(either plaques or colonies; see, for example, Sambrook).

Hybridization of such sequences may be carried out under stringentconditions. The term “stringent conditions” or “stringent hybridizationconditions” as used herein refers to conditions under which a probe willhybridize to its target sequence to a detectably greater degree than toother sequences (e.g., at least 2-fold, 5-fold, or 10-fold overbackground). Stringent conditions are sequence-dependent and will bedifferent in different circumstances. By controlling the stringency ofthe hybridization and/or washing conditions, target sequences that are100% complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 or 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a final wash in 0.1×SSC at 60 to 65° C. for at least about20 minutes. Optionally, wash buffers may comprise about 0.1% to about 1%SDS. The duration of hybridization is generally less than about 24hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) (thermal melting point)can be approximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, “% form” isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. Washes are typicallyperformed at least until equilibrium is reached and a low backgroundlevel of hybridization is achieved, such as for 2 hours, 1 hour, or 30minutes.

T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization, and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences with ≧90%identity are sought, the T_(m) can be decreased 10° C. Generally,stringent conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the T_(m);moderately stringent conditions can utilize a hybridization and/or washat 6, 7, 8, 9, or 10° C. lower than the T_(m); low stringency conditionscan utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C.lower than the T_(m).

Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), theSSC concentration can be increased so that a higher temperature can beused. An extensive guide to the hybridization of nucleic acids is foundin Tijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See also Sambrook. Thus, isolated sequences that encode a Cryprotein of the embodiments and hybridize under stringent conditions tothe Cry sequences disclosed herein, or to fragments thereof, areencompassed by the embodiments.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, as modified in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of theembodiments. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the embodiments. To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul et al. (1997) Nucleic AcidsRes. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See the National Center for Biotechnology Informationwebsite on the world wide web at ncbi.hlm.nih.gov. Alignment may also beperformed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. The term“equivalent program” as used herein refers to any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) supra, to find thealignment of two complete sequences that maximizes the number of matchesand minimizes the number of gaps. GAP considers all possible alignmentsand gap positions and creates the alignment with the largest number ofmatched bases and the fewest gaps. It allows for the provision of a gapcreation penalty and a gap extension penalty in units of matched bases.GAP must make a profit of gap creation penalty number of matches foreach gap it inserts. If a gap extension penalty greater than zero ischosen, GAP must, in addition, make a profit for each gap inserted ofthe length of the gap times the gap extension penalty. Default gapcreation penalty values and gap extension penalty values in Version 10of the GCG Wisconsin Genetics Software Package for protein sequences are8 and 2, respectively. For nucleotide sequences the default gap creationpenalty is 50 while the default gap extension penalty is 3. The gapcreation and gap extension penalties can be expressed as an integerselected from the group of integers consisting of from 0 to 200. Thus,for example, the gap creation and gap extension penalties can be 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%. 80%,90%, or 95% or more sequence identity when compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes generally means sequence identity of at least 60%, 70%, 80%,90%, or 95% or more sequence identity.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the T_(m) for the specific sequence at a defined ionic strength andpH. However, stringent conditions encompass temperatures in the range ofabout 1° C. to about 20° C. lower than the T_(m), depending upon thedesired degree of stringency as otherwise qualified herein. Nucleicacids that do not hybridize to each other under stringent conditions arestill substantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70%, 80%,85%, 90%, 95%, or more sequence identity to a reference sequence over aspecified comparison window. Optimal alignment for these purposes can beconducted using the global alignment algorithm of Needleman and Wunsch(1970) supra. An indication that two peptide sequences are substantiallyidentical is that one peptide is immunologically reactive withantibodies raised against the second peptide. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution. Peptides that are“substantially similar” share sequences as noted above except thatresidue positions that are not identical may differ by conservativeamino acid changes.

The use of the term “nucleotide constructs” herein is not intended tolimit the embodiments to nucleotide constructs comprising DNA. Those ofordinary skill in the art will recognize that nucleotide constructs,particularly polynucleotides and oligonucleotides composed ofribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides, may also be employed in the methods disclosedherein. The nucleotide constructs, nucleic acids, and nucleotidesequences of the embodiments additionally encompass all complementaryforms of such constructs, molecules, and sequences. Further, thenucleotide constructs, nucleotide molecules, and nucleotide sequences ofthe embodiments encompass all nucleotide constructs, molecules, andsequences which can be employed in the methods of the embodiments fortransforming plants including, but not limited to, those comprised ofdeoxyribonucleotides, ribonucleotides, and combinations thereof. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The nucleotide constructs,nucleic acids, and nucleotide sequences of the embodiments alsoencompass all forms of nucleotide constructs including, but not limitedto, single-stranded forms, double-stranded forms, hairpins,stem-and-loop structures, and the like.

A further embodiment relates to a transformed organism such as anorganism selected from the group consisting of plant and insect cells,bacteria, yeast, baculoviruses, protozoa, nematodes, and algae. Thetransformed organism comprises: a DNA molecule of the embodiments, anexpression cassette comprising the said DNA molecule, or a vectorcomprising the said expression cassette, which may be stablyincorporated into the genome of the transformed organism.

The sequences of the embodiments are provided in DNA constructs forexpression in the organism of interest. The construct will include 5′and 3′ regulatory sequences operably linked to a sequence of theembodiments. The term “operably linked” as used herein refers to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame. The construct may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multiple DNAconstructs.

Such a DNA construct is provided with a plurality of restriction sitesfor insertion of the Cry toxin sequence to be under the transcriptionalregulation of the regulatory regions. The DNA construct may additionallycontain selectable marker genes.

The DNA construct will include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the embodiments, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism serving as a host. Thetranscriptional initiation region (i.e., the promoter) may be native,analogous, foreign or heterologous to the host organism and/or to thesequence of the embodiments. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. The term“foreign” as used herein indicates that the promoter is not found in thenative organism into which the promoter is introduced. Where thepromoter is “foreign” or “heterologous” to the sequence of theembodiments, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked sequence of theembodiments. As used herein, a chimeric gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence. Where the promoter is a native ornatural sequence, the expression of the operably linked sequence isaltered from the wild-type expression, which results in an alteration inphenotype.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the sequence ofinterest, the plant host, or any combination thereof).

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, a nucleic acid may be optimized for increasedexpression in the host organism. Thus, where the host organism is aplant, the synthetic nucleic acids can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred codon usage. For example, although nucleic acid sequencesof the embodiments may be expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific codon preferences and GC content preferences ofmonocotyledons or dicotyledons as these preferences have been shown todiffer (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, themaize-preferred codon for a particular amino acid may be derived fromknown gene sequences from maize. Maize codon usage for 28 genes frommaize plants is listed in Table 4 of Murray et al., supra. Methods areavailable in the art for synthesizing plant-preferred genes. See, forexample, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al.(1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other well-characterized sequences that maybe deleterious to gene expression. The GC content of the sequence may beadjusted to levels average for a given cellular host, as calculated byreference to known genes expressed in the host cell. The term “hostcell” as used herein refers to a cell which contains a vector andsupports the replication and/or expression of the expression vector isintended. Host cells may be prokaryotic cells such as E. coli, oreukaryotic cells such as yeast, insect, amphibian, or mammalian cells,or monocotyledonous or dicotyledonous plant cells. An example of amonocotyledonous host cell is a maize host cell. When possible, thesequence is modified to avoid predicted hairpin secondary mRNAstructures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallieet al. (1995) Gene 165(2): 233-238), MDMV leader (Maize Dwarf MosaicVirus), human immunoglobulin heavy-chain binding protein (BiP) (Macejaket al. (1991) Nature 353: 90-94); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987)Nature 325: 622-625); tobacco mosaic virus leader (TMV) (Gallie et al.(1989) in Molecular Biology of RNA, ed. Cech (Liss, N.Y.), pp. 237-256);and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)Virology 81: 382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84: 965-968.

In preparing the expression cassette, the various DNA fragments may bemanipulated so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the embodiments.The promoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, inducible, orother promoters for expression in the host organism. Suitableconstitutive promoters for use in a plant host cell include, forexample, the core promoter of the Rsyn7 promoter and other constitutivepromoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the coreCaMV 35S promoter (Odell et al. (1985) Nature 313: 810-812); rice actin(McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen etal. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992)Plant Mol. Biol. 18: 675-689); pEMU (Last et al. (1991) Theor. Appl.Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALSpromoter (U.S. Pat. No. 5,659,026), and the like. Other constitutivepromoters include, for example, those discussed in U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142; and 6,177,611.

Depending on the desired outcome, it may be beneficial to express thegene from an inducible promoter. Of particular interest for regulatingthe expression of the nucleotide sequences of the embodiments in plantsare wound-inducible promoters. Such wound-inducible promoters, mayrespond to damage caused by insect feeding, and include potatoproteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology 14: 494-498); win1 andwin2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford et al. (1989)Mol. Gen. Genet. 215: 200-208); systemin (McGurl et al. (1992) Science225: 1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73-76); MPI gene(Corderok et al. (1994) Plant J. 6(2): 141-150); and the like, hereinincorporated by reference.

Additionally, pathogen-inducible promoters may be employed in themethods and nucleotide constructs of the embodiments. Suchpathogen-inducible promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. PlantPathol. 89: 245-254; Uknes et al. (1992) Plant Cell 4: 645-656; and VanLoon (1985) Plant Mol. Virol. 4: 111-116. See also WO 99/43819, hereinincorporated by reference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced pesticidalprotein expression within a particular plant tissue. Tissue-preferredpromoters include those discussed in Yamamoto et al. (1997) Plant J.12(2)255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803;Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al.(1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) PlantPhysiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524;Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994)Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant MolBiol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred or root-specific promoters are known and can be selectedfrom the many available from the literature or isolated de novo fromvarious compatible species. See, for example, Hire et al. (1992) PlantMol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetasegene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061(root-specific control element in the GRP 1.8 gene of French bean);Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specificpromoter of the mannopine synthase (MAS) gene of Agrobacteriumtumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22 (full-lengthcDNA clone encoding cytosolic glutamine synthetase (GS), which isexpressed in roots and root nodules of soybean). See also Bogusz et al.(1990) Plant Cell 2(7):633-641, where two root-specific promotersisolated from hemoglobin genes from the nitrogen-fixing nonlegumeParasponia andersonii and the related non-nitrogen-fixing nonlegumeTrema tomentosa are described. The promoters of these genes were linkedto a β-glucuronidase reporter gene and introduced into both thenonlegume Nicotiana tabacum and the legume Lotus corniculatus, and inboth instances root-specific promoter activity was preserved. Leach andAoyagi (1991) describe their analysis of the promoters of the highlyexpressed roIC and roID root-inducing genes of Agrobacterium rhizogenes(see Plant Science (Limerick) 79(1):69-76). They concluded that enhancerand tissue-preferred DNA determinants are dissociated in thosepromoters. Teeri et al. (1989) used gene fusion to lacZ to show that theAgrobacterium T-DNA gene encoding octopine synthase is especially activein the epidermis of the root tip and that the TR2′ gene is root specificin the intact plant and stimulated by wounding in leaf tissue, anespecially desirable combination of characteristics for use with aninsecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The TR1′gene fused to nptII (neomycin phosphotransferase II) showed similarcharacteristics. Additional root-preferred promoters include theVfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol.29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol.25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363;5,459,252; 5,401,836; 5,110,732; and 5,023,179.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); and milps(myo-inositol-1-phosphate synthase) (see U.S. Pat. No. 6,225,529, hereinincorporated by reference). Gamma-zein and Glob-1 are endosperm-specificpromoters. For dicots, seed-specific promoters include, but are notlimited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin,cruciferin, and the like. For monocots, seed-specific promoters include,but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein,g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO00/12733, where seed-preferred promoters from end1 and end2 genes aredisclosed; herein incorporated by reference. A promoter that has“preferred” expression in a particular tissue is expressed in thattissue to a greater degree than in at least one other plant tissue. Sometissue-preferred promoters show expression almost exclusively in theparticular tissue.

Where low level expression is desired, weak promoters will be used.Generally, the term “weak promoter” as used herein refers to a promoterthat drives expression of a coding sequence at a low level. By low levelexpression at levels of about 1/1000 transcripts to about 1/100,000transcripts to about 1/500,000 transcripts is intended. Alternatively,it is recognized that the term “weak promoters” also encompassespromoters that drive expression in only a few cells and not in others togive a total low level of expression. Where a promoter drives expressionat unacceptably high levels, portions of the promoter sequence can bedeleted or modified to decrease expression levels.

Such weak constitutive promoters include, for example the core promoterof the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, those disclosed in U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142; and 6,177,611; herein incorporated by reference.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitableselectable marker genes include, but are not limited to, genes encodingresistance to chloramphenicol (Herrera Estrella et al. (1983) EMBO J.2:987-992); methotrexate (Herrera Estrella et al. (1983) Nature303:209-213; and Meijer et al. (1991) Plant Mol. Biol. 16:807-820);streptomycin (Jones et al. (1987) Mol. Gen. Genet. 210:86-91);spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res.5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol. 7:171-176);sulfonamide (Guerineau et al. (1990) Plant Mol. Biol. 15:127-136);bromoxynil (Stalker et al. (1988) Science 242:419-423); glyphosate (Shawet al. (1986) Science 233:478-481; and U.S. application Ser. Nos.10/004,357; and 10/427,692); phosphinothricin (DeBlock et al. (1987)EMBO J. 6:2513-2518). See generally, Yarranton (1992) Curr. Opin.Biotech. 3: 506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci.USA 89: 6314-6318; Yao et al. (1992) Cell 71: 63-72; Reznikoff (1992)Mol. Microbiol. 6: 2419-2422; Barkley et al. (1980) in The Operon, pp.177-220; Hu et al. (1987) Cell 48: 555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52: 713-722; Deuschle et al. (1989)Proc. Natl. Acad. Sci. USA 86: 5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86: 2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines etal. (1993) Proc. Natl. Acad. Sci. USA 90: 1917-1921; Labow et al. (1990)Mol. Cell. Biol. 10: 3343-3356; Zambretti et al. (1992) Proc. Natl.Acad. Sci. USA 89: 3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci.USA 88: 5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104; Bonin(1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992)Proc. Natl. Acad. Sci. USA 89: 5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36: 913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); andGill et al. (1988) Nature 334: 721-724. Such disclosures are hereinincorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

The methods of the embodiments involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the embodiments do not depend on a particularmethod for introducing a polynucleotide or polypeptide into a plant,only that the polynucleotide or polypeptides gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotide or polypeptides into plants are known in the artincluding, but not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4: 320-334), electroporation(Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83: 5602-5606),Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al.(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al.(1988) Biotechnology 6: 923-926); and LecI transformation (WO 00/28058).For potato transformation see Tu et al. (1998) Plant Molecular Biology37: 829-838 and Chong et al. (2000) Transgenic Research 9: 71-78.Additional transformation procedures can be found in Weissinger et al.(1988) Ann. Rev. Genet. 22: 421-477; Sanford et al. (1987) ParticulateScience and Technology 5: 27-37 (onion); Christou et al. (1988) PlantPhysiol. 87: 671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8: 736-740 (rice);Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85: 4305-4309 (maize);Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos.5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol.91: 440-444 (maize); Fromm et al. (1990) Biotechnology 8: 833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc.Natl. Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al. (1985) inThe Experimental Manipulation of Ovule Tissues, ed. Chapman et al.(Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant CellReports 9: 415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) PlantCell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12: 250-255 and Christou and Ford (1995) Annals of Botany 75: 407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the sequences of the embodiments can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the Cry toxin protein or variants and fragmentsthereof directly into the plant or the introduction of the Cry toxintranscript into the plant. Such methods include, for example,microinjection or particle bombardment. See, for example, Crossway etal. (1986) Mol Gen. Genet. 202: 179-185; Nomura et al. (1986) Plant Sci.44: 53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 andHush et al. (1994) The Journal of Cell Science 107: 775-784, all ofwhich are herein incorporated by reference. Alternatively, the Cry toxinpolynucleotide can be transiently transformed into the plant usingtechniques known in the art. Such techniques include viral vector systemand the precipitation of the polynucleotide in a manner that precludessubsequent release of the DNA. Thus, transcription from theparticle-bound DNA can occur, but the frequency with which it isreleased to become integrated into the genome is greatly reduced. Suchmethods include the use of particles coated with polyethylimine (PEI;Sigma #P3143).

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the embodiments can be contained in transfercassette flanked by two non-identical recombination sites. The transfercassette is introduced into a plant have stably incorporated into itsgenome a target site which is flanked by two non-identical recombinationsites that correspond to the sites of the transfer cassette. Anappropriate recombinase is provided and the transfer cassette isintegrated at the target site. The polynucleotide of interest is therebyintegrated at a specific chromosomal position in the plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5: 81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive or inducible expression ofthe desired phenotypic characteristic identified. Two or moregenerations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited and thenseeds harvested to ensure that expression of the desired phenotypiccharacteristic has been achieved.

The nucleotide sequences of the embodiments may be provided to the plantby contacting the plant with a virus or viral nucleic acids. Generally,such methods involve incorporating the nucleotide construct of interestwithin a viral DNA or RNA molecule. It is recognized that therecombinant proteins of the embodiments may be initially synthesized aspart of a viral polyprotein, which later may be processed by proteolysisin vivo or in vitro to produce the desired pesticidal protein. It isalso recognized that such a viral polyprotein, comprising at least aportion of the amino acid sequence of a pesticidal protein of theembodiments, may have the desired pesticidal activity. Such viralpolyproteins and the nucleotide sequences that encode for them areencompassed by the embodiments. Methods for providing plants withnucleotide constructs and producing the encoded proteins in the plants,which involve viral DNA or RNA molecules are known in the art. See, forexample, U.S. Pat. Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367; and5,316,931; herein incorporated by reference.

The embodiments further relate to plant-propagating material of atransformed plant of the embodiments including, but not limited to,seeds, tubers, corms, bulbs, leaves, and cuttings of roots and shoots.

The embodiments may be used for transformation of any plant species,including, but not limited to, monocots and dicots. Examples of plantsof interest include, but are not limited to, corn (Zea mays), Brassicasp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the embodiments include, for example, pines suchas loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosapine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Montereypine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Westernhemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Plants of the embodiments include crop plants (for example, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Turfgrasses include, but are not limited to: annual bluegrass (Poaannua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poacompressa); Chewings fescue (Festuca rubra); colonial bentgrass(Agrostis tenuis); creeping bentgrass (Agrostis palustris); crestedwheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyroncristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poapratensis); orchardgrass (Dactylis glomerata); perennial ryegrass(Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba);rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smoothbromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy(Phleum pratense); velvet bentgrass (Agrostis canina); weepingalkaligrass (Puccinellia distans); western wheatgrass (Agropyronsmithii); Bermuda grass (Cynodon spp.); St. Augustine grass(Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass(Paspalum notatum); carpet grass (Axonopus affinis); centipede grass(Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum);seashore paspalum (Paspalum vaginatum); blue gramma (Boutelouagracilis); buffalo grass (Buchloe dactyloids); sideoats gramma(Bouteloua curtipendula).

Plants of interest include grain plants that provide seeds of interest,oil-seed plants, and leguminous plants. Seeds of interest include grainseeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc.Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica,maize, alfalfa, palm, coconut, flax, castor, olive etc. Leguminousplants include beans and peas. Beans include guar, locust bean,fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, favabean, lentils, chickpea, etc.

In certain embodiments the nucleic acid sequences of the embodiments canbe stacked with any combination of polynucleotide sequences of interestin order to create plants with a desired phenotype. For example, thepolynucleotides of the embodiments may be stacked with any otherpolynucleotides encoding polypeptides having pesticidal and/orinsecticidal activity, such as other Bt toxic proteins (described inU.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881;and Geiser et al. (1986) Gene 48:109), pentin (described in U.S. Pat.No. 5,981,722) and the like. The combinations generated can also includemultiple copies of any one of the polynucleotides of interest. Thepolynucleotides of the embodiments can also be stacked with any othergene or combination of genes to produce plants with a variety of desiredtrait combinations including but not limited to traits desirable foranimal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529);balanced amino acids (e.g. hordothionins (U.S. Pat. Nos. 5,990,389;5,885,801; 5,885,802; and 5,703,049); barley high lysine (Williamson etal. (1987) Eur. J. Biochem. 165: 99-106; and WO 98/20122) and highmethionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261: 6279;Kirihara et al. (1988) Gene 71: 359; and Musumura et al. (1989) PlantMol. Biol. 12: 123)); increased digestibility (e.g., modified storageproteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001); andthioredoxins (U.S. application Ser. No. 10/005,429, filed Dec. 3,2001)), the disclosures of which are herein incorporated by reference.

The polynucleotides of the embodiments can also be stacked with traitsdesirable for disease or herbicide resistance (e.g., fumonisindetoxification genes (U.S. Pat. No. 5,792,931); avirulence and diseaseresistance genes (Jones et al. (1994) Science 266:789; Martin et al.(1993) Science 262: 1432; and Mindrinos et al. (1994) Cell 78:1089);acetolactate synthase (ALS) mutants that lead to herbicide resistancesuch as the S4 and/or Hra mutations; inhibitors of glutamine synthasesuch as phosphinothricin or basta (e.g., bar gene); and glyphosateresistance (EPSPS gene and GAT gene as disclosed in U.S. applicationSer. Nos. 10/004,357; and 10/427,692); and traits desirable forprocessing or process products such as high oil (e.g., U.S. Pat. No.6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat.No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE) and starch debranching enzymes (SDBE)); and polymers orbioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert etal. (1988) J. Bacteriol. 170: 5837-5847) facilitate expression ofpolyhydroxyalkanoates (PHAs)), the disclosures of which are hereinincorporated by reference. One could also combine the polynucleotides ofthe embodiments with polynucleotides providing agronomic traits such asmale sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength,flowering time, or transformation technology traits such as cell cycleregulation or gene targeting (e.g. WO 99/61619; WO 00/17364; WO99/25821), the disclosures of which are herein incorporated byreference.

These stacked combinations can be created by any method including butnot limited to cross breeding plants by any conventional or TOPCROSS®methodology, or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference.

Compositions of the embodiments find use in protecting plants, seeds,and plant products in a variety of ways. For example, the compositionscan be used in a method that involves placing an effective amount of thepesticidal composition in the environment of the pest by a procedureselected from the group consisting of spraying, dusting, broadcasting,or seed coating.

Before plant propagation material (fruit, tuber, bulb, corm, grains,seed), but especially seed, is sold as a commercial product, it iscustomarily treated with a protectant coating comprising herbicides,insecticides, fungicides, bactericides, nematicides, molluscicides, ormixtures of several of these preparations, if desired together withfurther carriers, surfactants, or application-promoting adjuvantscustomarily employed in the art of formulation to provide protectionagainst damage caused by bacterial, fungal, or animal pests. In order totreat the seed, the protectant coating may be applied to the seedseither by impregnating the tubers or grains with a liquid formulation orby coating them with a combined wet or dry formulation. In addition, inspecial cases, other methods of application to plants are possible,e.g., treatment directed at the buds or the fruit.

The plant seed of the embodiments comprising a nucleotide sequenceencoding a pesticidal protein of the embodiments may be treated with aseed protectant coating comprising a seed treatment compound, such as,for example, captan, carboxin, thiram, methalaxyl, pirimiphos-methyl,and others that are commonly used in seed treatment. In one embodiment,a seed protectant coating comprising a pesticidal composition of theembodiments is used alone or in combination with one of the seedprotectant coatings customarily used in seed treatment.

It is recognized that the genes encoding the pesticidal proteins can beused to transform insect pathogenic organisms. Such organisms includebaculoviruses, fungi, protozoa, bacteria, and nematodes.

A gene encoding a pesticidal protein of the embodiments may beintroduced via a suitable vector into a microbial host, and said hostapplied to the environment, or to plants or animals. The term“introduced” in the context of inserting a nucleic acid into a cell,means “transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid into a eukaryotic orprokaryotic cell where the nucleic acid may be incorporated into thegenome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrialDNA), converted into an autonomous replicon, or transiently expressed(e.g., transfected mRNA).

Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one ormore crops of interest may be selected. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the pesticidalprotein, and desirably, provide for improved protection of the pesticidefrom environmental degradation and inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi,particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum,Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris,Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli andAzotobacter vinlandir and phytosphere yeast species such as Rhodotorularubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C.diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, andAureobasidium pollulans. Of particular interest are the pigmentedmicroorganisms.

A number of ways are available for introducing a gene expressing thepesticidal protein into the microorganism host under conditions thatallow for stable maintenance and expression of the gene. For example,expression cassettes can be constructed which include the nucleotideconstructs of interest operably linked with the transcriptional andtranslational regulatory signals for expression of the nucleotideconstructs, and a nucleotide sequence homologous with a sequence in thehost organism, whereby integration will occur, and/or a replicationsystem that is functional in the host, whereby integration or stablemaintenance will occur.

Transcriptional and translational regulatory signals include, but arenot limited to, promoters, transcriptional initiation start sites,operators, activators, enhancers, other regulatory elements, ribosomalbinding sites, an initiation codon, termination signals, and the like.See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2;Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed.Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.), hereinafter “Sambrook II”; Davis et al., eds. (1980)Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), ColdSpring Harbor, N.Y.; and the references cited therein.

Suitable host cells, where the pesticidal protein-containing cells willbe treated to prolong the activity of the pesticidal proteins in thecell when the treated cell is applied to the environment of the targetpest(s), may include either prokaryotes or eukaryotes, normally beinglimited to those cells that do not produce substances toxic to higherorganisms, such as mammals. However, organisms that produce substancestoxic to higher organisms could be used, where the toxin is unstable orthe level of application sufficiently low as to avoid any possibility oftoxicity to a mammalian host. As hosts, of particular interest will bethe prokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and gram-positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae.Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of pesticidal protein production include ease of introducingthe pesticidal protein gene into the host, availability of expressionsystems, efficiency of expression, stability of the protein in the host,and the presence of auxiliary genetic capabilities. Characteristics ofinterest for use as a pesticide microcapsule include protectivequalities for the pesticide, such as thick cell walls, pigmentation, andintracellular packaging or formation of inclusion bodies; leaf affinity;lack of mammalian toxicity; attractiveness to pests for ingestion; easeof killing and fixing without damage to the toxin; and the like. Otherconsiderations include ease of formulation and handling, economics,storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorulaspp., Aureobasidium spp., Saccharomyces spp. (such as S. cerevisiae),Sporobolomyces spp., phylloplane organisms such as Pseudomonas spp.(such as P. aeruginosa, P. fluorescens), Erwinia spp., andFlavobacterium spp., and other such organisms, including Bt, E. coli,Bacillus subtilis, and the like.

Genes encoding the pesticidal proteins of the embodiments can beintroduced into microorganisms that multiply on plants (epiphytes) todeliver pesticidal proteins to potential target pests. Epiphytes, forexample, can be gram-positive or gram-negative bacteria.

Root-colonizing bacteria, for example, can be isolated from the plant ofinterest by methods known in the art. Specifically, a Bacillus cereusstrain that colonizes roots can be isolated from roots of a plant (see,for example, Handelsman et al. (1991) Appl. Environ. Microbiol.56:713-718). Genes encoding the pesticidal proteins of the embodimentscan be introduced into a root-colonizing Bacillus cereus by standardmethods known in the art.

Genes encoding pesticidal proteins can be introduced, for example, intothe root-colonizing Bacillus by means of electrotransformation.Specifically, genes encoding the pesticidal proteins can be cloned intoa shuttle vector, for example, pHT3101 (Lerecius et al. (1989) FEMSMicrobiol. Letts. 60: 211-218. The shuttle vector pHT3101 containing thecoding sequence for the particular pesticidal protein gene can, forexample, be transformed into the root-colonizing Bacillus by means ofelectroporation (Lerecius et al. (1989) FEMS Microbiol. Letts. 60:211-218).

Expression systems can be designed so that pesticidal proteins aresecreted outside the cytoplasm of gram-negative bacteria, such as E.coli, for example. Advantages of having pesticidal proteins secretedare: (1) avoidance of potential cytotoxic effects of the pesticidalprotein expressed; and (2) improvement in the efficiency of purificationof the pesticidal protein, including, but not limited to, increasedefficiency in the recovery and purification of the protein per volumecell broth and decreased time and/or costs of recovery and purificationper unit protein.

Pesticidal proteins can be made to be secreted in E. coli, for example,by fusing an appropriate E. coli signal peptide to the amino-terminalend of the pesticidal protein. Signal peptides recognized by E. coli canbe found in proteins already known to be secreted in E. coli, forexample the OmpA protein (Ghrayeb et al. (1984) EMBO J, 3:2437-2442).OmpA is a major protein of the E. coli outer membrane, and thus itssignal peptide is thought to be efficient in the translocation process.Also, the OmpA signal peptide does not need to be modified beforeprocessing as may be the case for other signal peptides, for examplelipoprotein signal peptide (Duffaud et al. (1987) Meth. Enzymol. 153:492).

Pesticidal proteins of the embodiments can be fermented in a bacterialhost and the resulting bacteria processed and used as a microbial sprayin the same manner that Bt strains have been used as insecticidalsprays. In the case of a pesticidal protein(s) that is secreted fromBacillus, the secretion signal is removed or mutated using proceduresknown in the art. Such mutations and/or deletions prevent secretion ofthe pesticidal protein(s) into the growth medium during the fermentationprocess. The pesticidal proteins are retained within the cell, and thecells are then processed to yield the encapsulated pesticidal proteins.Any suitable microorganism can be used for this purpose. Pseudomonas hasbeen used to express Bt toxins as encapsulated proteins and theresulting cells processed and sprayed as an insecticide (Gaertner et al.(1993), in: Advanced Engineered Pesticides, ed. Kim).

Alternatively, the pesticidal proteins are produced by introducing aheterologous gene into a cellular host. Expression of the heterologousgene results, directly or indirectly, in the intracellular productionand maintenance of the pesticide. These cells are then treated underconditions that prolong the activity of the toxin produced in the cellwhen the cell is applied to the environment of target pest(s). Theresulting product retains the toxicity of the toxin. These naturallyencapsulated pesticidal proteins may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample EPA 0192319, and the references cited therein.

In the embodiments, a transformed microorganism (which includes wholeorganisms, cells, spore(s), pesticidal protein(s), pesticidalcomponent(s), pest-impacting component(s), mutant(s), living or deadcells and cell components, including mixtures of living and dead cellsand cell components, and including broken cells and cell components) oran isolated pesticidal protein can be formulated with an acceptablecarrier into a pesticidal composition(s) that is, for example, asuspension, a solution, an emulsion, a dusting powder, a dispersiblegranule or pellet, a wettable powder, and an emulsifiable concentrate,an aerosol or spray, an impregnated granule, an adjuvant, a coatablepaste, a colloid, and also encapsulations in, for example, polymersubstances. Such formulated compositions may be prepared by suchconventional means as desiccation, lyophilization, homogenization,extraction, filtration, centrifugation, sedimentation, or concentrationof a culture of cells comprising the polypeptide.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematicides,molluscicides, acaricides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular target pests.Suitable carriers and adjuvants can be solid or liquid and correspond tothe substances ordinarily employed in formulation technology, e.g.,natural or regenerated mineral substances, solvents, dispersants,wetting agents, tackifiers, binders, or fertilizers. The activeingredients of the embodiments are normally applied in the form ofcompositions and can be applied to the crop area, plant, or seed to betreated. For example, the compositions of the embodiments may be appliedto grain in preparation for or during storage in a grain bin or silo,etc. The compositions of the embodiments may be applied simultaneouslyor in succession with other compounds. Methods of applying an activeingredient of the embodiments or an agrochemical composition of theembodiments that contains at least one of the pesticidal proteinsproduced by the bacterial strains of the embodiments include, but arenot limited to, foliar application, seed coating, and soil application.The number of applications and the rate of application depend on theintensity of infestation by the corresponding pest.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; a carboxylateof a long chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate of dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include but are not limited to inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions of the embodiments can be in a suitable form for directapplication or as a concentrate of primary composition that requiresdilution with a suitable quantity of water or other diluent beforeapplication. The pesticidal concentration will vary depending upon thenature of the particular formulation, specifically, whether it is aconcentrate or to be used directly. The composition contains 1 to 98% ofa solid or liquid inert carrier, and 0 to 50% or 0.1 to 50% of asurfactant. These compositions will be administered at the labeled ratefor the commercial product, for example, about 0.01 lb-5.0 lb. per acrewhen in dry form and at about 0.01 pts.-10 pts. per acre when in liquidform.

In a further embodiment, the compositions, as well as the transformedmicroorganisms and pesticidal proteins of the embodiments, can betreated prior to formulation to prolong the pesticidal activity whenapplied to the environment of a target pest as long as the pretreatmentis not deleterious to the pesticidal activity. Such treatment can be bychemical and/or physical means as long as the treatment does notdeleteriously affect the properties of the composition(s). Examples ofchemical reagents include but are not limited to halogenating agents;aldehydes such as formaldehyde and glutaraldehyde; anti-infectives, suchas zephiran chloride; alcohols, such as isopropanol and ethanol; andhistological fixatives, such as Bouin's fixative and Helly's fixative(see, for example, Humason (1967) Animal Tissue Techniques (W.H. Freemanand Co.).

In other embodiments, it may be advantageous to treat the Cry toxinpolypeptides with a protease, for example trypsin, to activate theprotein prior to application of a pesticidal protein composition of theembodiments to the environment of the target pest. Methods for theactivation of protoxin by a serine protease are well known in the art.See, for example, Cooksey (1968) Biochem. J. 6:445-454 and Carroll andEllar (1989) Biochem. J. 261:99-105, the teachings of which are hereinincorporated by reference. For example, a suitable activation protocolincludes, but is not limited to, combining a polypeptide to beactivated, for example a purified novel Cry polypeptide (e.g., havingthe amino acid sequence set forth in SEQ ID NO:2), and trypsin at a1/100 weight ratio of protein/trypsin in 20 nM NaHCO₃, pH 8 anddigesting the sample at 36° C. for 3 hours.

The compositions (including the transformed microorganisms andpesticidal proteins of the embodiments) can be applied to theenvironment of an insect pest by, for example, spraying, atomizing,dusting, scattering, coating or pouring, introducing into or on thesoil, introducing into irrigation water, by seed treatment or generalapplication or dusting at the time when the pest has begun to appear orbefore the appearance of pests as a protective measure. For example, thepesticidal protein and/or transformed microorganisms of the embodimentsmay be mixed with grain to protect the grain during storage. It isgenerally important to obtain good control of pests in the early stagesof plant growth, as this is the time when the plant can be most severelydamaged. The compositions of the embodiments can conveniently containanother insecticide if this is thought necessary. In one embodiment, thecomposition is applied directly to the soil, at a time of planting, ingranular form of a composition of a carrier and dead cells of a Bacillusstrain or transformed microorganism of the embodiments. Anotherembodiment is a granular form of a composition comprising anagrochemical such as, for example, an herbicide, an insecticide, afertilizer, an inert carrier, and dead cells of a Bacillus strain ortransformed microorganism of the embodiments.

Those skilled in the art will recognize that not all compounds areequally effective against all pests. Compounds of the embodimentsdisplay activity against insect pests, which may include economicallyimportant agronomic, forest, greenhouse, nursery, ornamentals, food andfiber, public and animal health, domestic and commercial structure,household, and stored product pests. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyLepidoptera.

Larvae and adults of the order Coleoptera include weevils from thefamilies Anthribidae, Bruchidae, and Curculionidae (including, but notlimited to: Anthonomus grandis Boheman (boll weevil); Lissorhoptrusoryzophilus Kuschel (rice water weevil); Sitophilus granarius Linnaeus(granary weevil); S. oryzae Linnaeus (rice weevil); Hypera punctataFabricius (clover leaf weevil); Cylindrocopturus adspersus LeConte(sunflower stem weevil); Smicronyx fulvus LeConte (red sunflower seedweevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorusmaidis Chittenden (maize billbug)); flea beetles, cucumber beetles,rootworms, leaf beetles, potato beetles, and leafminers in the familyChrysomelidae (including, but not limited to: Leptinotarsa decemlineataSay (Colorado potato beetle); Diabrotica virgifera virgifera LeConte(western corn rootworm); D. barberi Smith & Lawrence (northern cornrootworm); D. undecimpunctata howardi Barber (southern corn rootworm);Chaetocnema pulicaria Melsheimer (corn flea beetle); Phyllotretacruciferae Goeze (corn flea beetle); Colaspis brunnea Fabricius (grapecolaspis); Oulema melanopus Linnaeus (cereal leaf beetle); Zygogrammaexclamationis Fabricius (sunflower beetle)); beetles from the familyCoccinellidae (including, but not limited to: Epilachna varivestisMulsant (Mexican bean beetle)); chafers and other beetles from thefamily Scarabaeidae (including, but not limited to: Popillia japonicaNewman (Japanese beetle); Cyclocephala borealis Arrow (northern maskedchafer, white grub); C. immaculata Olivier (southern masked chafer,white grub); Rhizotrogus majalis Razoumowsky (European chafer);Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer(carrot beetle)); carpet beetles from the family Dermestidae; wirewormsfrom the family Elateridae, Eleodes spp., Melanotus spp.; Conoderusspp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; barkbeetles from the family Scolytidae and beetles from the familyTenebrionidae.

Larvae of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers, and heliothines in the family NoctuidaeSpodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beetarmyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar);Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogoniaMorrison (western cutworm); A. subterranea Fabricius (granulatecutworm); Alabama argillacea Hübner (cotton leaf worm); Trichoplusia niHübner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabraFabricius (green cloverworm); Heliothis virescens Fabricius (tobaccobudworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindaraBarnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris(darksided cutworm); Earias insulana Boisduval (spiny bollworm); E.vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner(American bollworm); H. zea Boddie (corn earworm or cotton bollworm);Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialisGrote (citrus cutworm); borers, casebearers, webworms, coneworms, andskeletonizers from the family Pyralidae Ostrinia nubilalis Hübner(European corn borer); Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautellaWalker (almond moth); Chilo suppressalis Walker (rice stem borer); C.partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm); C. teterrellusZincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leafroller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinataLinnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraeagrandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius(surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestiaelutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker (sod webworm);Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellusZeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalisWalker (tea tree web moth); Maruca testulalis Geyer (bean pod borer);Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulasWalker (yellow stem borer); Udea rubigalis Guenée (celery leaftier); andleafrollers, budworms, seed worms, and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Archips argyrospilaWalker (fruit tree leaf roller); A. rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischer vonRösslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham(banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.pomonella Linnaeus (coding moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Lobesia botrana Denis & Schiffermüller (European grape vine moth);Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth);Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguellaHübner (vine moth); Bonagota salubricola Meyrick (Brazilian appleleafroller); Grapholita molesta Busck (oriental fruit moth); Suleimahelianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneuraspp.

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith(orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese OakSilkmoth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfacaterpillar); Datana integerrima Grote & Robinson (walnut caterpillar);Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomossubsignaria Hübner (elm spanworm); Erannis tiliaria Harris (lindenlooper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisinaamericana Guérin-Méneville (grapeleaf skeletonizer); Hemileuca oliviaeCockrell (range caterpillar); Hyphantria cunea Drury (fall webworm);Keiferia lycopersicella Walsingham (tomato pinworm); Lambdinafiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellarialugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus(satin moth); Lymantria dispar Linnaeus (gypsy moth); Manducaquinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M.sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumataLinnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm);Papilio cresphontes Cramer (giant swallowtail, orange dog); Phryganidiacalifornica Packard (California oakworm); Phyllocnistis citrellaStainton (citrus leafminer); Phyllonorycter blancardella Fabricius(spotted tentiform leafminer); Pieris brassicae Linnaeus (large whitebutterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus(green veined white butterfly); Platyptilia carduidactyla Riley(artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth);Pectinophora gossypiella Saunders (pink bollworm); Pontia protodiceBoisduval & Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée(omnivorous looper); Schizura concinna J. E. Smith (red humpedcaterpillar); Sitotroga cerealella Olivier (Angoumois grain moth);Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar);Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick(tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothissubflexa Guenée; Malacosoma spp. and Orgyia spp.

Adults and immatures of the order Diptera include: leafminers such asAgromyza parvicornis Loew (corn blotch leafminer); midges (including,but not limited to: Contarinia sorghicola Coquillett (sorghum midge);Mayetiola destructor Say (Hessian fly); Sitodiplosis mosellana Géhin(wheat midge); Neolasioptera murtfeldtiana Felt, (sunflower seedmidge)); fruit flies (Tephritidae), Oscinella frit Linnaeus (fritflies); maggots (including, but not limited to: Delia platura Meigen(seedcorn maggot); D. coarctata Fallen (wheat bulb fly); and other Deliaspp., Meromyza americana Fitch (wheat stem maggot); Musca domesticaLinnaeus (house flies); Fannia canicularis Linnaeus, F. femoralis Stein(lesser house flies); Stomoxys calcitrans Linnaeus (stable flies)); faceflies, horn flies, blow flies, Chrysomya spp.; Phormia spp.; and othermuscoid fly pests, horse flies Tabanus spp.; bot flies Gastrophilusspp.; Oestrus spp.; cattle grubs Hypoderma spp.; deer flies Chrysopsspp.; Melophagus ovinus Linnaeus (keds); and other Brachycera,mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; black fliesProsimulium spp.; Simulium spp.; biting midges, sand flies, sciarids,and other Nematocera.

Adults and nymphs of the orders Hemiptera and Homoptera include insectssuch as, but not limited to, adelgids from the family Adelgidae, plantbugs from the family Miridae, cicadas from the family Cicadidae,leafhoppers, Empoasca spp.; from the family Cicadellidae, planthoppersfrom the families Cixiidae, Flatidae, Fulgoroidea, Issidae andDelphacidae, treehoppers from the family Membracidae, psyllids from thefamily Psyllidae, whiteflies from the family Aleyrodidae, aphids fromthe family Aphididae, phylloxera from the family Phylloxeridae,mealybugs from the family Pseudococcidae, scales from the familiesAsterolecanidae, Coccidae, Dactylopiidae, Diaspididae, Eriococcidae,Ortheziidae, Phoenicococcidae and Margarodidae, lace bugs from thefamily Tingidae, stink bugs from the family Pentatomidae, cinch bugs,Blissus spp.; and other seed bugs from the family Lygaeidae, spittlebugsfrom the family Cercopidae squash bugs from the family Coreidae, and redbugs and cotton stainers from the family Pyrrhocoridae.

Agronomically important members from the order Homoptera furtherinclude, but are not limited to: Acyrthisiphon pisum Harris (pea aphid);Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black beanaphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicisForbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantagineaPaaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly appleaphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopteruspruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnipaphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphumeuphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potatoaphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch(corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphisgraminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcaneaphid); Sitobion avenae Fabricius (English grain aphid); Therioaphismaculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer deFonscolombe (black citrus aphid); and T. citricida Kirkaldy (browncitrus aphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande(pecan phylloxera); Bemisia tabaci Gennadius (tobacco whitefly,sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleafwhitefly); Dialeurodes citri Ashmead (citrus whitefly); Trialeurodesabutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood(greenhouse whitefly); Empoasca fabae Harris (potato leafhopper);Laodelphax striatellus Fallen (smaller brown planthopper); Macrolestesquadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler(green leafhopper); N. nigropictus Stål (rice leafhopper); Nilaparvatalugens Stål (brown planthopper); Peregrinus maidis Ashmead (cornplanthopper); Sogatella furcifera Horvath (white-backed planthopper);Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee(white apple leafhopper); Erythroneoura spp. (grape leafhoppers);Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasiMaskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock(San Jose scale); Planococcus citri Risso (citrus mealybug);Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster(pear psylla); Trioza diospyri Ashmead (persimmon psylla).

Agronomically important species from the order Hemiptera include, butare not limited to: Acrosternum hilare Say (green stink bug); Anasatristis De Geer (squash bug); Blissus leucopterus leucopterus Say(chinch bug); Corythuca gossypii Fabricius (cotton lace bug);Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellusHerrich-Schäffer (cotton stainer); Euschistus servus Say (brown stinkbug); E. variolarius Palisot de Beauvois (one-spotted stink bug);Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say(leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois(tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug);L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius(European tarnished plant bug); Lygocoris pabulinus Linnaeus (commongreen capsid); Nezara viridula Linnaeus (southern green stink bug);Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas(large milkweed bug); Pseudatomoscelis seriatus Reuter (cottonfleahopper).

Insects included in the order Hemiptera include: Calocoris norvegicusGmelin (strawberry bug); Orthops campestris Linnaeus; Plesiocorisrugicollis Fallen (apple capsid); Cyrtopeltis modestus Distant (tomatobug); Cyrtopeltis notatus Distant (suckfly); Spanagonicus albofasciatusReuter (whitemarked fleahopper); Diaphnocoris chlorionis Say(honeylocust plant bug); Labopidicola allii Knight (onion plant bug);Pseudatomoscelis seriatus Reuter (cotton flea hopper); Adelphocorisrapidus Say (rapid plant bug); Poecilocapsus lineatus Fabricius(four-lined plant bug); Nysius ericae Schilling (false chinch bug);Nysius raphanus Howard (false chinch bug); Nezara viridula Linnaeus(Southern green stink bug); Eurygaster spp.; Coreidae spp.;Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp.;and Cimicidae spp.

Adults and larvae of the order Acari (mites) include: Aceria tosichellaKeifer (wheat curl mite); Petrobia latens Müller (brown wheat mite);spider mites and red mites in the family Tetranychidae, Panonychus ulmiKoch (European red mite); Tetranychus urticae Koch (two spotted spidermite); (T. mcdanieli McGregor (McDaniel mite); T. cinnabarinus Boisduval(carmine spider mite); T. turkestani Ugarov & Nikolski (strawberryspider mite); flat mites in the family Tenuipalpidae, Brevipalpus lewisiMcGregor (citrus flat mite); rust and bud mites in the familyEriophyidae and other foliar feeding mites and mites important in humanand animal health, i.e. dust mites in the family Epidermoptidae,follicle mites in the family Demodicidae, grain mites in the familyGlycyphagidae, ticks in the order Ixodidae. Ixodes scapularis Say (deertick); I. holocyclus Neumann (Australian paralysis tick); Dermacentorvariabilis Say (American dog tick); Amblyomma americanum Linnaeus (lonestar tick); and scab and itch mites in the families Psoroptidae,Pyemotidae, and Sarcoptidae.

Insect pests of the order Thysanura include Lepisma saccharina Linnaeus(silverfish); Thermobia domestica Packard (firebrat). Additionalarthropod pests include: spiders in the order Araneae such as Loxoscelesreclusa Gertsch & Mulaik (brown recluse spider); and the Latrodectusmactans Fabricius (black widow spider); and centipedes in the orderScutigeromorpha such as Scutigera coleoptrata Linnaeus (housecentipede).

Insect pests may be tested for pesticidal activity of compositions ofthe embodiments in early developmental stages, e.g., as larvae or otherimmature forms. The insects may be reared in total darkness at fromabout 20° C. to about 30° C. and from about 30% to about 70% relativehumidity. Bioassays may be performed as described in Czapla and Lang(1990) J. Econ. Entomol. 83(6): 2480-2485. Methods of rearing insectlarvae and performing bioassays are well known to one of ordinary skillin the art.

A wide variety of bioassay techniques are known to one skilled in theart. General procedures include addition of the experimental compound ororganism to the diet source in an enclosed container. Pesticidalactivity can be measured by, but is not limited to, changes inmortality, weight loss, attraction, repellency and other behavioral andphysical changes after feeding and exposure for an appropriate length oftime. Bioassays described herein can be used with any feeding insectpest in the larval or adult stage.

The following examples are presented by way of illustration, not by wayof limitation.

EXPERIMENTAL Example 1 Bioassay for Testing the Pesticidal Activity ofthe B. thuringiensis Toxin Against Selected Insects

Bioassays were conducted to evaluate the effects of the Bt insecticidaltoxin peptide, set forth in SEQ ID NO: 2, on Western corn rootworm.Feeding assays were conducted on an artificial diet containing theinsecticidal protein. The insecticidal protein was topically appliedusing a coleopteran-specific artificial diet. The toxin was applied at arate of 1.0 μg per 25 μL sample per well and allowed to dry. The proteinis in 10 mM carbonate buffer at a pH of 10. One neonate larva was placedin each well to feed ad libitum for 5 days. Results were expressed aspositive for larvae reactions such as stunting and or mortality. Resultswere expressed as negative if the larvae were similar to the negativecontrol that is feeding diet to which the above buffer only has beenapplied.

TABLE 1 Results of feeding bioassay for SEQ ID NO: 2 Insect TestedResult Western corn rootworm (Diabrotica virgifera) +

Example 2 Determination of LC₅₀ and EC₅₀

Bioassays are conducted to determine an LC₅₀ and EC₅₀ of theinsecticidal toxin peptide, set forth in SEQ ID NO: 2, on Western cornrootworm (Diabrotica virgifera). Feeding assays are conducted on anartificial diet containing the insecticidal protein. The insecticidalprotein is diluted with 10 mM carbonate buffer at pH 10 and with insectdiet to give a final toxin concentration of 50000, 5000, 500, 50 and 5ppm. One neonate larva is placed in each well to feed ad libitum for 5days. Each bioassay is done with eight duplicates at each dose and thebioassay is replicated three times. Results are expressed as LC₅₀ formortality and/or EC₅₀ by weighing the surviving larvae at each toxinconcentration.

Example 3 Transformation of Maize by Particle Bombardment andRegeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with aDNA molecule containing the toxin nucleotide sequence (e.g., SEQ IDNO: 1) operably linked to a ubiquitin promoter and the selectable markergene PAT (Wohlleben et al. (1988) Gene 70: 25-37), which confersresistance to the herbicide Bialaphos. Alternatively, the selectablemarker gene is provided on a separate DNA molecule. Transformation isperformed as follows. Media recipes follow below.

Preparation of Target Tissue

The ears are husked and surface sterilized in 30% CLOROX™ bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5 cm target zone in preparation forbombardment.

Preparation of DNA

A plasmid vector comprising a toxin nucleotide sequence (e.g., SEQ IDNO: 1) operably linked to a ubiquitin promoter is made. For example, asuitable transformation vector comprises a UBI1 promoter from Zea mays,a 5′ UTR from UBI1 and a UBI1 intron, in combination with a PinIIterminator. The vector additionally contains a PAT selectable markergene driven by a CAMV35S promoter and includes a CAMV35S terminator.Optionally, the selectable marker can reside on a separate plasmid. ADNA molecule comprising a toxin nucleotide sequence as well as a PATselectable marker is precipitated onto 1.1 μm (average diameter)tungsten pellets using a CaCl₂ precipitation procedure as follows:

-   -   100 μL prepared tungsten particles in water    -   10 μL (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)    -   100 μL 2.5 M CaC1₂    -   10 μL 0.1 M spermidine

Each reagent is added sequentially to a tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 mL 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μL 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μLspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for expression of the toxin by assaysknown in the art or as described above.

Bombardment and Culture Media

Bombardment medium (560Y) comprises 4.0 g/L N6 basal salts (SIGMAC-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/Lthiamine HCl, 120.0 g/L sucrose, 1.0 mg/L 2,4-D, and 2.88 g/L L-proline(brought to volume with dl H₂0 following adjustment to pH 5.8 with KOH);2.0 g/L Gelrite™ (added after bringing to volume with dl H₂0); and 8.5mg/L silver nitrate (added after sterilizing the medium and cooling toroom temperature). Selection medium (560R) comprises 4.0 g/L N6 basalsalts (SIGMA C-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0 g/L sucrose, and 2.0 mg/L 2,4-D(brought to volume with dl H₂0 following adjustment to pH 5.8 with KOH);3.0 g/L Gelrite™ (added after bringing to volume with dl H₂0); and 0.85mg/L silver nitrate and 3.0 mg/L Bialaphos (both added after sterilizingthe medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/L MS salts (GIBCO11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/L thiamine HCl, 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycinebrought to volume with polished D-I H₂0) (Murashige and Skoog (1962)Physiol. Plant. 15: 473), 100 mg/L myo-inositol, 0.5 mg/L zeatin, 60 g/Lsucrose, and 1.0 mL/L of 0.1 mM abscisic acid (brought to volume withpolished dl H₂0 after adjusting to pH 5.6); 3.0 g/L Gelrite™ (addedafter bringing to volume with dl H₂0); and 1.0 mg/L indoleacetic acidand 3.0 mg/L Bialaphos (added after sterilizing the medium and coolingto 60° C.).

Hormone-free medium (272V) comprises 4.3 g/L MS salts (GIBCO 11117-074),5.0 mL/L MS vitamins stock solution (0.100 g/L nicotinic acid, 0.02 g/Lthiamine HCl, 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycine brought tovolume with polished dl H₂0), 0.1 g/L myo-inositol, and 40.0 g/L sucrose(brought to volume with polished dl H₂0 after adjusting pH to 5.6); and6 g/L Bacto-agar (added after bringing to volume with polished dl H₂0),sterilized and cooled to 60° C.

Example 4 Agrobacterium-Mediated Transformation of Maize andRegeneration of Transgenic Plants

For Agrobacterium-mediated transformation of maize with a toxinnucleotide sequence (e.g., SEQ ID NO: 1), the method of Zhao can be used(U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326; thecontents of which are hereby incorporated by reference). Briefly,immature embryos are isolated from maize and the embryos contacted witha suspension of Agrobacterium under conditions whereby the bacteria arecapable of transferring the toxin nucleotide sequence (SEQ ID NO: 1) toat least one cell of at least one of the immature embryos (step 1: theinfection step). In this step the immature embryos can be immersed in anAgrobacterium suspension for the initiation of inoculation. The embryosare co-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). The immature embryos can be cultured on solidmedium following the infection step. Following this co-cultivationperiod an optional “resting” step is contemplated. In this resting step,the embryos are incubated in the presence of at least one antibioticknown to inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants (step 3: resting step). Theimmature embryos can be cultured on solid medium with antibiotic, butwithout a selecting agent, for elimination of Agrobacterium and for aresting phase for the infected cells. Next, inoculated embryos arecultured on medium containing a selective agent and growing transformedcallus is recovered (step 4: the selection step). The immature embryosare cultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and calli grown onselective medium can be cultured on solid medium to regenerate theplants.

Example 5 Transformation of Soybean Embryos

Soybean embryos are bombarded with a plasmid containing the toxinnucleotide sequence of SEQ ID NO: 1 operably linked to a pinII promoteras follows. To induce somatic embryos, cotyledons, 3-5 mm in lengthdissected from surface-sterilized, immature seeds of an appropriatesoybean cultivar are cultured in the light or dark at 26° C. on anappropriate agar medium for six to ten weeks. Somatic embryos producingsecondary embryos are then excised and placed into a suitable liquidmedium. After repeated selection for clusters of somatic embryos thatmultiplied as early, globular-staged embryos, the suspensions aremaintained as described below.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327: 70-73, U.S. Pat. No. 4,945,050). A Du Pont Biolistic PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313: 810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25: 179-188), and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising an toxin nucleotidesequence (e.g., SEQ ID NO: 1) operably linked to the pinII promoter canbe isolated as a restriction fragment. This fragment can then beinserted into a unique restriction site of the vector carrying themarker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post-bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the embodiments.

That which is claimed:
 1. An isolated nucleic acid molecule wherein saidnucleic acid molecule encodes a polypeptide sequence having insecticidalactivity selected from the group consisting of: a) a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1; and b) anucleic acid molecule which encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:2, wherein the nucleic acid molecule encodesa polypeptide having insecticidal activity.
 2. The isolated nucleic acidmolecule of claim 1, wherein said nucleic acid molecule is a syntheticsequence that has been designed for expression in a plant.
 3. A DNAconstruct comprising the nucleic acid molecule of claim
 1. 4. The DNAconstruct of claim 3, further comprising a nucleic acid moleculeencoding a heterologous polypeptide.
 5. A host cell that contains theDNA construct of claim
 3. 6. The host cell of claim 5 that is abacterial cell.
 7. The host cell of claim 5 that is a plant cell.
 8. Atransgenic plant comprising the host cell of claim
 7. 9. The transgenicplant of claim 8, wherein said plant is selected from the groupconsisting of maize, sorghum, wheat, cabbage, sunflower, tomato,crucifers, peppers, potato, cotton, rice, soybean, sugar beet,sugarcane, tobacco, barley, and oilseed rape.
 10. A transformed seed ofthe plant of claim 9, wherein the seed comprises the DNA construct. 11.A method for producing a polypeptide with insecticidal activity, saidmethod comprising culturing the host cell of claim 5 under conditions inwhich a nucleic acid molecule encoding the polypeptide is expressed,said polypeptide being selected from the group consisting of: a) apolypeptide comprising the amino acid sequence of SEQ ID NO:2; and b) apolypeptide that is encoded by the nucleotide sequence of SEQ ID NO:1.12. A plant having stably incorporated into its genome a DNA constructcomprising a nucleic acid molecule that encodes a protein havinginsecticidal activity, wherein said nucleic acid molecule is selectedfrom the group consisting of: a) a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1; and b) a nucleic acid molecule whichencodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2wherein said nucleotide sequence nucleic acid molecule is operablylinked to a promoter that drives expression of a coding sequence in aplant cell.
 13. A method for protecting a plant from a pest, said methodcomprising introducing into said plant or cell thereof at least oneexpression vector comprising a nucleic acid molecule that encodes aninsecticidal polypeptide, wherein said nucleic acid molecule is selectedfrom the group consisting of: a) a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1; and b) a nucleic acid molecule whichencodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2wherein the nucleic acid molecule encodes a polypeptide havinginsecticidal activity.
 14. The method of claim 13, wherein said plantproduces an insecticidal polypeptide having insecticidal activityagainst a coleopteran pest.